fluorescence-tagged antibodies with anti-mouse efluor 450-conjugated cd45  (Thermo Fisher)

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Anti-HIV-1 HSPC-based gene therapy with safety kill switch to defend against and attack HIV-1 infection

doi: 10.1016/j.omtm.2025.101486

Figure Lengend Snippet: Efficient human HSPC vector-modification, transplantation, and multi-lineage human hematopoietic cell reconstitution in huBLT mice (A) Experimental design for the investigation of M1 and U1 vectors in NSG huBLT mice. Human FL-CD34 + cells were transduced with M1 or U1 vectors at MOI 3 on day −1. NSG huBLT mice were conditioned with 270 cGy of sub-lethal body irradiation from a cesium-137 source. Mice were transplanted with the vector-transduced FL-CD34 + HSPC and human thymus tissue on day 0. Mice were challenged with R5-tropic HIV-1 NFNSXSL9 (200 ng p24/mouse) at 11 weeks post-transplant. (B) huEGFRt and D1D2CAR 4-1BB transgene expression in vector-transduced FL-CD34 + cells in ex-vivo culture. huEGFRt and D1D2CAR 4-1BB co-expressing population was determined by mAb staining and flow cytometry 4 days post-vector transduction. (C) Human multilineage hematopoietic cell reconstitution in peripheral blood from 8 weeks post-vector-transduced HSPC transplant. Cell surface markers of human lymphocytes (CD45), T cells (CD3, CD4, and CD8), and B cells (CD19) were determined by mAb staining and flow cytometry. Dots and error bars show mean ± SEM, respectively. (D) Vector-marking levels were determined in peripheral blood cells from 8 weeks to 17 weeks post-transplant by digital PCR. Average vector DNA copies were calculated by VCN = (WPRE DNA copies in vector DNA/ul)/(human β-globin copies/ul/2). Dots and error bars show mean ± SEM, respectively. (E) Human multilineage hematopoietic cell expansion within huEGFRt-expressing population after HIV-1 infection. Expression of huEGFRt was determined by mAb staining and flow cytometry. Cell surface markers of human lymphocytes (CD45), T cells (CD3, CD4, and CD8), and B cells (CD19) were also determined by mAb staining and flow cytometry and gated within the huEGFRt + population. Mice were challenged with HIV-1 at 11 weeks-post transplant (not noted in this figure). Dots and error bars show mean ± SEM, respectively. Mann-Whitney U test was performed to calculate significance, ∗ p < 0.05. (F) Ex vivo cytokine production measured by cytokine release assay. CD8 + T splenocytes from M1 huBLT mice were co-cultured with Env+ target cells (PMA/ionomycin-activated ACH2 cells) or unstimulated Env− cells (medium only) as a negative control ex vivo . Data were collected from our replicate huBLT mice experiment (donor 2). Cells were collected at time of mouse sacrifice at week 20 post-transplant. Cytokine expression was measured by flow cytometry. Dots and error bars show mean ± SEM, respectively. t test with Holm-Šídák adjustment was performed to calculate significance. ∗ p < 0.05. (G) Viral loads were measured as HIV-1 RNA copies per mL in mouse plasma every 2 weeks post- HIV-1 challenge by digital PCR in 2 different sets of experiments using 2 human CD34 + HSPC donors (donor 1 and donor 2). huBLT mice groups were transplanted with either M1- ( n = 5 in both experiments) or U1-transduced ( n = 7) HSPC. Untransduced huBLT mice were used as a negative control in both experiments ( n = 3 in first experiment, and n = 5 in replicate experiment). Data were shown as mean ± SEM. t test with Holm-Šídák adjustment was performed to calculate significance. ns, not significant, ∗ p < 0.05 and ∗∗∗ p < 0.001.

Article Snippet: The following antibodies were used: CD45-eFluor 450 (HI30, eBioscience), CD3-APC H7 (SK7, Pharmingen), CD4-APC (OKT4, eBioscience), CD8-PerCP Cy5.5 (SK1, BioLegend), CD19-Brilliant Violet 605 (HIB19, BD Horizon), EGFR-PE (Hu1, R&D Systems), and Countbright beads (Invitrogen).

Techniques: Plasmid Preparation, Modification, Transplantation Assay, Transduction, Irradiation, Expressing, Ex Vivo, Staining, Flow Cytometry, Digital PCR, Infection, MANN-WHITNEY, Release Assay, Cell Culture, Negative Control, Clinical Proteomics

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Anti-HIV-1 HSPC-based gene therapy with safety kill switch to defend against and attack HIV-1 infection

doi: 10.1016/j.omtm.2025.101486

Figure Lengend Snippet: Cetuximab-mediated negative selection of huEGFRt expressing vector-modified human hematopoietic cells in huBLT mice (A) Experimental design for the investigation of CTX-mediated negative selection of huEGFRt + vector-modified human cells. CTX treatment group M1 huBLT mice ( n = 5) were injected with 1 mg per mouse intraperitoneally for 11 consecutive days. Human natural killer (NK) cells (5 × 10 6 ) per mouse were injected retro-orbitally (RO) one day before first treatment (D0) and on D7 during CTX treatment. IL-15 expressing lentiviral vectors (2.5 × 10 6 IU) were injected RO on day 7 of CTX treatment (2.5 × 10 5 IU/mouse). CTX untreated group ( n = 4) served as a negative control. (B) huEGFRt expression level in multilineage human peripheral blood cells (CD45 + , CD3 + , CD19 + , CD4 + , and CD8 + ) in CTX-treated and -untreated M1 huBLT mice. Blood was collected 1 week before CTX treatment, and 1 and 4 weeks post-onset of CTX treatment. Samples were stained with mAbs and measured by flow cytometry. huEGFRt expression was measured by flow cytometry using mAb CTX-PE. Dots and error bars show mean ± SEM, respectively. Mann-Whitney U test was performed to calculate significance. ns, not significant, and ∗∗ p < 0.01. (C) Absolute multilineage huEGFRt + cell count in CTX-treated and -untreated M1 huBLT mice in peripheral blood. Blood was collected at weeks 13, 15, and 18 post-transplantation (1 week pre-CTX treatment, and 1 and 4 weeks post-CTX treatment, respectively). huEGFRt and surface markers of human lymphocytes (CD45), T cells (CD3, CD4, and CD8), and B cells (CD19), were stained by mAbs and measured by flow cytometry. Mann-Whitney U test was performed to calculate significance. ns, not significant, ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. (D) huEGFRt expression level across multilineage human T cell populations (CD45 + , CD3 + , CD4 + , CD8 + , and CD4 + CD8 + ) in spleen and bone marrow tissue collected from CTX-treated and -untreated M1 huBLT mice. Samples were stained with mAb CTX-PE. Dots and error bars show mean ± SEM, respectively. t test with Holm-Šídák adjustment was performed to calculate significance. ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001. (E) Representative flow cytometry data showing huEGFRt expression within CD34 + /CD90 + /CD38 − population of bone marrow cells in CTX-treated and -untreated M1 huBLT mice. Samples were stained with mAb CTX-PE and measured by flow cytometry. (F) Cumulative data showing huEGFRt expression within CD34 + /CD90 + /CD38 − population of bone marrow cells collected from CTX-treated and -untreated M1 huBLT mice. Samples were stained with mAb CTX-PE and measured by flow cytometry. %huEGFRt was normalized to background levels from mock transduced HSPC transplanted huBLT mice. Dots and error bars show mean ± SEM, respectively. Student’s t test was performed to calculate significance. ∗ p < 0.05.

Article Snippet: The following antibodies were used: CD45-eFluor 450 (HI30, eBioscience), CD3-APC H7 (SK7, Pharmingen), CD4-APC (OKT4, eBioscience), CD8-PerCP Cy5.5 (SK1, BioLegend), CD19-Brilliant Violet 605 (HIB19, BD Horizon), EGFR-PE (Hu1, R&D Systems), and Countbright beads (Invitrogen).

Techniques: Selection, Expressing, Plasmid Preparation, Modification, Injection, Negative Control, Staining, Flow Cytometry, MANN-WHITNEY, Cell Counting, Transplantation Assay