invivomab rat anti-mouse cd4 igg2b clone gk1.5  (Bio X Cell)

Journal: bioRxiv

Article Title: NK cell immunotherapy administered at the time of HIV recrudescence is associated with viral control

doi: 10.1101/2025.11.19.688897

Figure Lengend Snippet: A) Schematic figure showing the overview of NK cell production and the overview of the mouse study timeline. Red circles on the timeline indicate blood collection, black dots indicate scheduled necropsies. Schematic of transgene design: MND promoter, CAR (CD4-MBL-CD8TM-4-1BB-CD3z), CXCR5, IL-15, the BgH PolyA tail, with separation mediated by P2A and T2A sites, and inverted terminal repeats (ITRs). Schematic showing expression of all transgene components as well as base editor-mediated knock out of PD-1 on the NK cell surface. Figure made using Biorender.com. B) Expression of CAR (MBL) and CXCR5 molecules was confirmed in the cell product using flow cytometry. Cells were pre-gated sequentially on lymphocytes, singlets, live cells, CD56+, and CD3-. C) PD-1 knockout was detected at the RNA level by RT-PCR, CAR NK (blue) relative to control NK cells (black). D) IL-15 expression in CAR NK (blue) and control NK (black) cells via IL-15 ELISA of cell culture supernatant. E) CAR (blue) and control (black) cell proliferation over one week post-thaw. F) The specific lysis of HIV Envelope P815s by CAR (blue) or control (black) NK cells via DELFIA cytotoxicity assay. 5:1, 10:1, and 20:1 E:T ratios were tested. All assays were performed in duplicate or triplicate. Data are represented as mean + standard deviation (error bars).

Article Snippet: After a 2-day recovery, CAR NK cells were sorted on CD56+ CD4+ using the MACSQuant Tyto Cell Sorter (Miltenyi Biotec) and expanded for an additional 7 days following the procedure described above.

Techniques: Expressing, Knock-Out, Flow Cytometry, Reverse Transcription Polymerase Chain Reaction, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Lysis, Cytotoxicity Assay, Standard Deviation

Journal: bioRxiv

Article Title: NK cell immunotherapy administered at the time of HIV recrudescence is associated with viral control

doi: 10.1101/2025.11.19.688897

Figure Lengend Snippet: NK (CD56+) cells in CAR (blue), control (black), or PBS (red) treated groups from A) Peripheral Blood, B) Lymph node (at necropsies), and C) Spleen (at necropsies). CAR+CXCR5+ NK cells over time in D) Peripheral Blood, E) Lymph node (at necropsies), and F) Spleen (at necropsies). Flow was pre-gated on Lymphocytes, Singlets, Live Cells, human CD45+, mouse CD45-, CD3+, and then through CD56+ MBL+ CXCR5+ (see Fig. S4 for gating strategy). Median viral loads (green), NK cell levels (orange), post-treatment in G) controllers or H) non-controllers. I) Median human hematopoietic cell counts (CD45+)/ ul blood of controllers (gray) or non-controllers (pink).

Article Snippet: After a 2-day recovery, CAR NK cells were sorted on CD56+ CD4+ using the MACSQuant Tyto Cell Sorter (Miltenyi Biotec) and expanded for an additional 7 days following the procedure described above.

Techniques: Control

Journal: bioRxiv

Article Title: NK cell immunotherapy administered at the time of HIV recrudescence is associated with viral control

doi: 10.1101/2025.11.19.688897

Figure Lengend Snippet: CD4:CD8 ratios over time in A) CAR NK-treated animals (blue), B) control NK-treated animals (black), and C) PBS-treated animals (red). CD4:CD8 ratios in CAR (blue), control NK (black), and PBS (red) treated groups at D) -6 DPT, E) 6 DPT, F) 14 DPT, G) 28 DPT, H) 42 DPT, and I) 56 DPT. Lines represent median values. CD4:CD8 ratios were determined by flow cytometry and pre-gated on Lymphocytes, Singlets, Live Cells, Human CD45+, Mouse CD45-, CD3+ (see gating strategy in Fig. S4 ).

Article Snippet: After a 2-day recovery, CAR NK cells were sorted on CD56+ CD4+ using the MACSQuant Tyto Cell Sorter (Miltenyi Biotec) and expanded for an additional 7 days following the procedure described above.

Techniques: Control, Flow Cytometry

Journal: Animal Cells and Systems

Article Title: Novel TLR2 agonist Amuc_C derived from Akkermansia muciniphila exhibits potent anti-tumor activity in colorectal cancers

doi: 10.1080/19768354.2025.2578019

Figure Lengend Snippet: The immunofluorescence staining of CT26 tumors harvested from control and Amuc_C-treated groups. (A) T cell panel. CD3, CD8, and CD4 were stained in green, red, and pink; (B) M1 macrophage panel. CD86 and iNOS were stained pink and red; (C) M2 macrophage panel. CD206 and arginase-1 (Arg-1) were stained in red and pink. Cell nuclei were stained with DAPI. (scale bar = 20 μm). Images were quantified randomly in five fields at 20x magnification. All data are presented as mean ± SEM. Statistical significance was calculated by one-way ANOVA with the Tukey test for the comparisons between all groups (ns, not significant; *, p < 0.05; ****, p < 0.0001).

Article Snippet: The following monoclonal antibodies were used to assess the phenotypes of T lymphocytes, macrophages, and dendritic cells: PE/Cyanine7 anti-mouse CD45 (BioLegend, clone 30-F11, Cat. No. 103114), FITC anti-mouse Ly-6C (BioLegend, clone HK1.4, Cat. No. 128006), rat anti-Mouse CD3: FITC (Bio–Rad, Hercules, CA, USA, clone KT3, Cat. No. MCA500F), rat anti-Mouse CD4: RPE (Bio–Rad, clone RM4-5, Cat. No. MCA2691PE), rat anti-Mouse CD8: Alexa Fluor® 647 (Bio–-Rad, clone YTS169.4, Cat. No. MCA1768A647), PE anti-mouse F4/80 (BioLegend, clone BM8, Cat. No. 123110), APC/Cyanine7 anti-mouse/human CD11b (BioLegend, clone M1/70, Cat. No. 101226), PerCP/Cyanine5.5 anti-mouse I-A/I-E (BioLegend, clone M5/114.15.2, Cat. No. 107626), APC anti-mouse CD11c (BioLegend, clone N418, Cat. No. 117310), and PE anti-mouse CD103 (BioLegend, clone 2E7, Cat. No. 121406).

Techniques: Immunofluorescence, Staining, Control

Journal: Veterinary Sciences

Article Title: Evaluation of PD-L1 and TIM-3 Pathways in T Cells During Experimental Bovine Leukemia Virus Infection in Sheep

doi: 10.3390/vetsci12090810

Figure Lengend Snippet: Figure 1. Cross-reactivity of anti-bovine PD-L1 and TIM-3 monoclonal antibodies in ovine PBMCs. (A) Cross-reactivity of anti-bovine PD-L1 monoclonal antibody (mAb) with fresh ovine PBMCs. The gray histogram shows fluorescence intensity of the rat IgG2a isotype control. The pink histogram shows staining with anti-bovine PD-L1 mAb. All data were gated on live IgM+ cells within PBMCs. (B–D) Cross-reactivity of anti-bovine TIM-3 mAb with fresh ovine PBMCs. The gray histograms show fluorescence intensity of the mouse IgG1 isotype control. The red histograms show staining with anti-bovine TIM-3 mAb. Histograms were gated on CD4+ (B), CD8+ (C), and γδTCR+ T cells (D). Gating strategies for the flow cytometric assays were shown in Supplementary Figure S1.

Article Snippet: Cells were then washed and stained with Alexa Fluor 647-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific), Alexa Fluor 488-labeled anti-ovine CD4 mAb (17D; mouse IgG1; Washington State University Monoclonal Antibody Center, Pullman, WA, USA), phycoerythrin (PE)-conjugated anti-bovine CD8 mAb (CC63; mouse IgG1; Bio-Rad, Hercules, CA, USA), PE/cyanine7 (PE/Cy7)-conjugated anti-ovine γδTCR mAb (86D; mouse IgG1; Washington State University Monoclonal Antibody Center), and Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific) at 4 ◦C for 20 min.

Techniques: Bioprocessing, Fluorescence, Control, Staining

Journal: Veterinary Sciences

Article Title: Evaluation of PD-L1 and TIM-3 Pathways in T Cells During Experimental Bovine Leukemia Virus Infection in Sheep

doi: 10.3390/vetsci12090810

Figure Lengend Snippet: Figure 2. Blockade effects of anti-bovine PD-L1 and TIM-3 monoclonal antibodies in PBMCs of healthy sheep. (A–C) T-cell functional analysis in PBMCs from BLV-uninfected healthy sheep (n = 10) with blockade by anti-PD-L1 and anti-TIM-3 mAbs under ConA stimulation. (A) Fre- quency of activated T cells (CD25+CD69+) within CD4+ and CD8+ T-cell subsets. (B,C) Frequency of cytokine-producing activated cells (CD69+IFN-γ+ and CD69+TNF-α+) within CD4+ and CD8+ T-cell subsets. Gating strategies for the flow cytometric assays are shown in Supplementary Figures S2 and S3. Each symbol represents data from an individual animal. Red lines indicate median values. Significant differences compared between treatment groups: * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Cells were then washed and stained with Alexa Fluor 647-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific), Alexa Fluor 488-labeled anti-ovine CD4 mAb (17D; mouse IgG1; Washington State University Monoclonal Antibody Center, Pullman, WA, USA), phycoerythrin (PE)-conjugated anti-bovine CD8 mAb (CC63; mouse IgG1; Bio-Rad, Hercules, CA, USA), PE/cyanine7 (PE/Cy7)-conjugated anti-ovine γδTCR mAb (86D; mouse IgG1; Washington State University Monoclonal Antibody Center), and Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific) at 4 ◦C for 20 min.

Techniques: Bioprocessing, Functional Assay

Journal: Veterinary Sciences

Article Title: Evaluation of PD-L1 and TIM-3 Pathways in T Cells During Experimental Bovine Leukemia Virus Infection in Sheep

doi: 10.3390/vetsci12090810

Figure Lengend Snippet: Figure 4. Comparison of immunoinhibitory receptor expression between healthy and experimentally BLV-infected sheep. (A) Percentage of PD-L1+ cells among IgM+ B cells in BLV-uninfected (n = 4) and BLV-infected sheep (n = 7). (B–D) Comparison of TIM-3+ cell frequencies in CD4+, CD8+, and γδTCR+

Article Snippet: Cells were then washed and stained with Alexa Fluor 647-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific), Alexa Fluor 488-labeled anti-ovine CD4 mAb (17D; mouse IgG1; Washington State University Monoclonal Antibody Center, Pullman, WA, USA), phycoerythrin (PE)-conjugated anti-bovine CD8 mAb (CC63; mouse IgG1; Bio-Rad, Hercules, CA, USA), PE/cyanine7 (PE/Cy7)-conjugated anti-ovine γδTCR mAb (86D; mouse IgG1; Washington State University Monoclonal Antibody Center), and Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific) at 4 ◦C for 20 min.

Techniques: Comparison, Expressing, Infection

Journal: Veterinary Sciences

Article Title: Evaluation of PD-L1 and TIM-3 Pathways in T Cells During Experimental Bovine Leukemia Virus Infection in Sheep

doi: 10.3390/vetsci12090810

Figure Lengend Snippet: Figure 5. T-cell activation and cytokine responses in BLV-infected sheep following immune checkpoint blockade. (A,B) Frequencies of activated CD25+CD69+ cells in CD4+ and CD8+ T-cell subsets in PBMCs from BLV-infected sheep (n = 7) following ConA stimulation with blockade by anti-PD-L1 and anti-TIM-3 mAbs. (C–F) Frequencies of cytokine-producing CD69+ cells expressing IFN-γ (C,D) or TNF-α (E,F) under the same blockade conditions. (G) IFN-γ concentration of PBMCs from BLV- infected sheep (n = 7) following ConA stimulation with blockade by anti-PD-L1 and anti-TIM-3 mAbs. Gating strategies for the flow cytometric assays are shown in Supplementary Figures S2 and S3. Each dot represents data from an individual animal. Red lines indicate median values. Significant differences compared between treatment groups: * p < 0.05, ** p < 0.01.

Article Snippet: Cells were then washed and stained with Alexa Fluor 647-conjugated anti-mouse IgG antibody (Thermo Fisher Scientific), Alexa Fluor 488-labeled anti-ovine CD4 mAb (17D; mouse IgG1; Washington State University Monoclonal Antibody Center, Pullman, WA, USA), phycoerythrin (PE)-conjugated anti-bovine CD8 mAb (CC63; mouse IgG1; Bio-Rad, Hercules, CA, USA), PE/cyanine7 (PE/Cy7)-conjugated anti-ovine γδTCR mAb (86D; mouse IgG1; Washington State University Monoclonal Antibody Center), and Fixable Viability Dye eFluor 780 (Thermo Fisher Scientific) at 4 ◦C for 20 min.

Techniques: Activation Assay, Infection, Expressing, Concentration Assay

Journal: iScience

Article Title: Photoconverted cells allow rapid assessment of vaccine adjuvant potency in mice

doi: 10.1016/j.isci.2025.112774

Figure Lengend Snippet: Induction of anti-tumor immune responses by various adjuvanted OVA (A–E) Schematic of representation of the experimental design. Naive female C57BL/six mice were inoculated s.c. with E.G7-OVA cells (5∗10 5 ) in the right flank. Once tumor reached to approximately 20 mm 3 , CR108, MF59, Alum plus OVA, and OVA alone were administrated near the tumor sites, respectively, with PBS as a vehicle control from days 1, 4, and 7 (A). Average growth curves shown are mean ± SEM from two independent experiments (PBS, n = 8; OVA, n = 8; Alum + OVA, n = 8; MF59 + OVA, n = 8; CR108 + OVA, n = 9) (B). Statistical significances: # (CR108 + OVA vs. Alum + OVA, p < 0.05), ##(CR108 + OVA vs. Alum + OVA, p < 0.01) ∗(CR108 + OVA vs. MF59 + OVA, p < 0.05), and ns (CR108 + OVA vs. MF59 + OVA, not significant) by Student’s t test (C). The tumor weights were determined for each group. Quantification of tumor-infiltrating CD45 + CD3 + CD8 + T cells (D), and CD45 + CD3 + CD4 + T cells were determined by FACS at the end of the experiment (E) ( A). Data shown are representative of two independent experiments with four mice in each experiment (mean ± SEM). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by one-way ANOVA (C, D, E). (F) In vivo CTL response measurement: C57BL/six mice were subcutaneously immunized with CR108 + OVA, MF59 + OVA, Alum + OVA, OVA alone, or PBS on days 0 and 14. On day 21 post-first immunization. Cells pulsed with OVA 257–264 peptides were stained with CFSE high , while unpulsed cells were stained with CFSE low . CFSE high , and CFSE low cells were mixed in 1:1 and then intravenously transferred into the immunized mice. Twenty hours later, splenocytes were analyzed by flow cytometry to assess OVA-specific lysis. Data shown are representative of two independent experiments with 5–6 mice in each experiment (PBS, n = 6; OVA, n = 6; Alum + OVA, n = 6; MF59 + OVA, n = 6; CR108 + OVA, n = 5) (mean ± SEM). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by One-way ANOVA. (G–I) Schematic representation of the treatment experimental design. Naive female C57BL/6 mice were inoculated s.c. with E.G7-OVA cells (5∗10 5 ) in the right flank. Once tumor reached approximately 20 mm 3 , CR108, MF59, Alum plus OVA, and OVA alone were administrated near the tumor sites, respectively, with PBS as a vehicle control from days 1, 7, and 14 (G). Average tumor growth curves (H) and the survival rates were measured until day 17 (I). Data shown are mean ± SEM from two independent experiments (untreated, n = 9; OVA, n = 10; Alum + OVA, n = 9; MF59 + OVA, n = 10; CR108 + OVA, n = 9). ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by two-way ANOVA (H). (J–L)Experimental design for α-CD4 or α-CD8 blocking. naive female C57BL/six mice were inoculated s.c. with 5 × 10 5 E.G7-OVA cells in the right flank. Once tumors reached 20 mm 3 , mice were intraperitoneally (i.p.) treated with 100 μg α-CD4 (GK1.5, BioXcell), 100 μg α-CD8 (2.43, BioXcell), or 100 μg rat IgG2b isotype control (LTF-2, BioXcell) on days 0, 5, 10, and 15. CR108 + OVA or PBS (untreated control) was administered near the tumor site on days 1, 7, and 13 (J). Average tumor growth curves (K) and survival rates (L) were determined. Data shown are mean ± SEM from two independent experiments (α-CD8 CR108 + OVA, n = 7; α-CD4 CR108 + OVA, n = 8; isotype CR108 + OVA, n = 7). Statistical significance: ∗∗∗∗ p < 0.0001, ∗∗∗ p < 0.001, ∗∗ p < 0.01, ∗ p < 0.05, ns (not significant) by two-way ANOVA (K).

Article Snippet: InVivoMAb Rat anti-mouse CD4 IgG2b; Clone GK1.5 , BioXcell, West Lafayette, IN, USA , Cat# BE0003-1, RRID: AB_1107636.

Techniques: Control, In Vivo, Staining, Flow Cytometry, Lysis, Blocking Assay

Journal: iScience

Article Title: Photoconverted cells allow rapid assessment of vaccine adjuvant potency in mice

doi: 10.1016/j.isci.2025.112774

Figure Lengend Snippet: Loss of CD8 + T cell activation due to CCR7 blockage affecting Mig DCs recruitment (A–C) Experimental procedure: the hair-clipped skin of KikGR mice was exposed to 436 nm violet light at an intensity of 400 mW/cm 2 for 4 min. Three hours later, 10 μg of α-CCR7 (4B12, R&D) blocking antibody per mouse was injected subcutaneously at the photoconverted skin site, while 10 μg of rat IgG2a antibody (2A3, BioXcell) per mouse was used as an isotype control. Six hours later, CR108 or MF59 plus OVA were administered subcutaneously at the α-CCR7 injection site. Forty-eight hours after administration, the numbers of (A) MHCII hi CD11c med Mig DCs, (B) KikGR-red cells, and (C) MHCII med CD11c hi Res DCs in the dLNs were measured by FACS. Data shown are representative of two independent experiments with three mice in each experiment (mean ± SEM). ∗ p < 0.05, ns (not significant) by Student’s t test (A, B, C). (D–H) Schematic of Experimental Design: CFSE-labeled OTI CD8 + T cells (1 × 10 6 ) were transferred into naive C57BL/six mice. One day later, the mice were subcutaneously injected with 10 μg of α-CCR7 per mouse or 10 μg of rat IgG2a antibody per mouse as an isotype control. Subsequently, the mice were immunized at the antibody-injected site with CR108 or MF59 plus OVA, and PBS as a vehicle control. Three days post-immunization, CFSE lo CD45.1 + CD3 + CD8 + TCRVα2 + T cells in dLNs, non-dLNs, and spleens were analyzed by FACS (D). Histogram analysis: histogram overlays of CFSE-dilution in CD45.1 + TCRVα2 + CD8 + T cells from dLNs, non-dLNs, and spleens were presented. (E) Quantitative Analysis: The number of CFSE lo CD45.1 + TCRVα2 + CD8 + T cells in dLNs (F), non-dLNs (G), and spleens (H) was quantified. Data shown are representative of two independent experiments with 3–4 mice in each experiment (mean ± SEM) (isotype CR108 + OVA, n = 4; α-CCR7 CR108 + OVA, n = 4; isotype MF59 + OVA, n = 3; α-CCR7 MF59 + OVA, n = 4; PBS, n = 4). ∗∗∗∗ p < 0.0001; ∗∗∗ p < 0.001; ∗∗ p < 0.01; ∗ p < 0.05; ns, not significant by Student’s t test (F, G, H). (I) Experimental design for α-CCR7 blocking: naive female C57BL/six mice were inoculated subcutaneously with E.G7-OVA cells (1 × 10 6 ) in the right flank, and tumor growth was monitored until the volume reached 20 mm 3 . To block Mig DCs migration, mice bearing E.G7-OVA tumors were treated subcutaneously with 10 μg of α-CCR7 per mouse or 10 μg of rat IgG2a antibody per mouse as an isotype control on days 1, 7, and 13. CR108 plus OVA were administered near the tumor sites, and PBS as a vehicle control (untreated), on days 1, 7, and 13. Tumor Growth Curves: Average tumor growth curves (α-CCR7 CR108 + OVA, n = 5; isotype CR108 + OVA, n = 4) were determined. Data shown are mean ± SEM from two independent experiments. ∗ p < 0.05 by two-way ANOVA.

Article Snippet: InVivoMAb Rat anti-mouse CD4 IgG2b; Clone GK1.5 , BioXcell, West Lafayette, IN, USA , Cat# BE0003-1, RRID: AB_1107636.

Techniques: Activation Assay, Blocking Assay, Injection, Control, Labeling, Migration