Journal: Nature Communications

Article Title: Unveiling the cut-and-repair cycle of designer nucleases in human stem and T cells via CLEAR-time dPCR

doi: 10.1038/s41467-025-65182-4

Figure Lengend Snippet: a Schematic of wildtype and large deletion cassettes used to establish the LoD of large deletions, and representation of clonal expansion of cells harbouring a large deletion (orange) mediated genotoxic aberration amongst wildtype cells (blue). Dark and light grey bar indicates CCR5 and reference sequences, respectively. Red bar indicates flag sequence used to fuse assay and reference sequences. b Correlation of observed against the expected percentage of large deletions. Vertical black dotted line represents the limit of detection. Solid and dotted red line represents line of regression with 95% confidence interval, respectively. R 2 = 0.997. Calculated with linear regression analysis in GraphPad. Data shown as mean ± s.d. of n = 4 technical replicates. c CLEAR-time dPCR summaries of Cas9-edited HSPCs targeting various genes at different timepoints. The BTK edited HSPCs were also transduced with AAV6 encoding GFP. All editing was normalised against unedited mock electroporated HSPCs. Data are shown as mean ± s.d. d CLEAR-time dPCR summary of SH2D1A edited T cells with Cas9, Cas12, and TALENs at 3 days post-editing. Data are shown as mean ± s.d. e CLEAR-time dPCR summary of on-target CCR5 edited with decreasing concentrations of Cas9 at 3 h and 3 days post-editing. Data are shown as mean ± s.d. f DSB and indels quantification at three known off-targets targeting CCR5 with decreasing concentrations of Cas9 at 3 h and 3 days post-editing. Data are shown as mean ± s.d. g CLEAR-time dPCR on XIAP edited HSPCs pre-transplant and 16 weeks post-transplant. Data are shown as mean ± s.d. n = 4 mice for each treatment group. h Integration frequency in 16-week post-transplant XIAP edited and AAV-transduced hCD45 cells by dPCR and flow cytometry. Flow cytometry data represent n = 1 per mouse, dPCR data shown as mean ± s.d. of n = 3 technical replicates. i CLEAR-time dPCR normalised ICE analysis of pre-transplant XIAP edited HSPCs and post-transplant XIAP edited hCD45 cells. All data represents n = 3 technical replicates unless stated otherwise. d – f Two-way ANOVA with Tukey post-hoc test. n.s.= no statistical significance, **** p < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Human CD45-positive cells were positively enriched using hCD45 microbeads (Miltenyi Biotec) and MS columns (Miltenyi Biotec) following manufacturer instructions.

Techniques: Sequencing, Transduction, TALENs, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Tumor-dependent myeloid and lymphoid cell recruitment in genO-BRGSF-HIS mice: a novel tool for evaluating immunotherapies

doi: 10.3389/fimmu.2025.1624724

Figure Lengend Snippet: genO-BRGSF-HIS mice develop stable lymphoid, myeloid, and dendritic compartments for 31 weeks, and the myeloid and dendritic compartments can be restimulated. (A) The rate of humanization was measured in the blood over 31 weeks (w) in genO-BRGSF-HIS mice (n=22) engrafted with hCD34 + cells from two donors (black circles and white circles). Percentage of hCD45 + cells relative to total live leukocytes is shown; horizontal lines represent the mean. (B) Quantification of the different cell populations following hCD34 + HSC engraftment over 31 weeks, represented as a percentage relative to human CD45 + cells; horizontal lines represent the mean. Data in (A, B) were analyzed using a Mann Whitney test. ****P<0.0001.

Article Snippet: For splenocyte stimulation, mouse CD45 + cells were depleted and hCD45 + -enriched cells were then stimulated with either CpG ODN 2216 (Invivogen #tlrl-2216) at 10 μM (for stimulation of TLR9); Poly(I:C) (Invivogen # tlrl-picw) at 10 μg/mL (for stimulation of TLR3), R837 (Invivogen # tlrl-imqs) at 10 μg/mL (for stimulation of TLR7); R848 (Resiquimod, Invivogen, #tlrl-r848) at 10 μg/mL (for stimulation of TLR7/8) or 2’3’ cGAMP (Invivogen # tlrl-nacga23) at 10 μg/mL (for stimulation of STING).

Techniques: MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: Tumor-dependent myeloid and lymphoid cell recruitment in genO-BRGSF-HIS mice: a novel tool for evaluating immunotherapies

doi: 10.3389/fimmu.2025.1624724

Figure Lengend Snippet: genO-BRGSF-HIS mice develop different subsets of lymphoid cells. (A) Quantification of the B cell subpopulations in the spleen, bone marrow (BM), and blood of hFlt3L-treated genO-BRGSF-HIS mice at 19 weeks of age (n=5, 3 hCD34 + cell donors). Quantifications are represented in percentages relative to hCD45 + cells. (B) Quantification of the T cell subpopulations in the spleen or bone marrow (BM) of genO-BRGSF-HIS mice at 18 weeks of age (n=13, 6 hCD34 + cell donors), represented in percentages relative to hCD45 + cells. Error bars represent standard deviation individual points represent individual mice.

Article Snippet: For splenocyte stimulation, mouse CD45 + cells were depleted and hCD45 + -enriched cells were then stimulated with either CpG ODN 2216 (Invivogen #tlrl-2216) at 10 μM (for stimulation of TLR9); Poly(I:C) (Invivogen # tlrl-picw) at 10 μg/mL (for stimulation of TLR3), R837 (Invivogen # tlrl-imqs) at 10 μg/mL (for stimulation of TLR7); R848 (Resiquimod, Invivogen, #tlrl-r848) at 10 μg/mL (for stimulation of TLR7/8) or 2’3’ cGAMP (Invivogen # tlrl-nacga23) at 10 μg/mL (for stimulation of STING).

Techniques: Standard Deviation

Journal: Frontiers in Immunology

Article Title: Tumor-dependent myeloid and lymphoid cell recruitment in genO-BRGSF-HIS mice: a novel tool for evaluating immunotherapies

doi: 10.3389/fimmu.2025.1624724

Figure Lengend Snippet: The MDA-MB-231 TME is mainly composed of myeloid cells, and its composition is dependent on tumor burden. (A) Tumor growth was measured over time in hFlt3L-treated genO-BRGSF-HIS mice engrafted with hCD34 + cells and implanted with 10 x 10 6 MDA-MB-231 cells. (B) Mouse body weight was quantified following implantation with 10 x 10 6 MDA-MB-231 cells in hFlt3L-treated genO-BRGSF-HIS mice. Mouse body weight is represented per hCD34 + HSC donor. For graphs (A, B) , five mice were used per donor (25 mice in total). (C) Quantification of the percentage of murine or human CD45 + cells as part of the immune cell infiltrate in MDA-MB-231 tumors at an average size of 200, 500 or 800 mm 3 . Mean percentages are plotted. (D) Quantification of the cell populations present in the human immune infiltrate of the MDA-MB-231 TME at an average size of 200, 500 or 800 mm 3 by cytometry. Mean percentages are plotted. For (C, D) , the graphs represent tumors in 19 hFlt3L-treated genO-BRGSF-HIS mice, engrafted with hCD34 + cells from three donors and implanted with 10 x 10 6 tumor cells. Six mice were used in the 200 mm 3 timepoint (2 mice/donor), nine for the 500 mm 3 timepoint (3 mice/donor) and four mice for the 800 mm 3 timepoint (1 mouse for donors 1 and 2, two mice for donor 3). (E) Quantification of activated M2-like macrophages (CD80 + /CD86 + ) within the M2-like population (CD206 + /CD163 + ). (F) Percentage of cells expressing ILT2 and ILT4 among the M2-like macrophage population (CD206 + /CD163 + ). Graphs (E, F) represent tumors in 24 hFlt3L-treated genO-BRGSF-HIS mice. (G) Percentage of cells expressing PD-L1 among the population of M1-like (CD206 - /CD163 - /CD86 + ) and M2-like macrophages (CD206 + /CD163 + ). (H) Mean percentage of cells expressing PD-1 among the population of Tregs and conventional CD4 T cells in tumors of 400–600 mm 3 . Graphs (G, H) represent tumors in 8 hFlt3L-treated genO-BRGSF-HIS mice. In panels (E–H) , data was acquired in tumors of 400–600 mm 3 , individual points represent individual mice, and the different colors represent different donors (five in total for E, F and three for G, H ). Error bars represent standard deviation.

Article Snippet: For splenocyte stimulation, mouse CD45 + cells were depleted and hCD45 + -enriched cells were then stimulated with either CpG ODN 2216 (Invivogen #tlrl-2216) at 10 μM (for stimulation of TLR9); Poly(I:C) (Invivogen # tlrl-picw) at 10 μg/mL (for stimulation of TLR3), R837 (Invivogen # tlrl-imqs) at 10 μg/mL (for stimulation of TLR7); R848 (Resiquimod, Invivogen, #tlrl-r848) at 10 μg/mL (for stimulation of TLR7/8) or 2’3’ cGAMP (Invivogen # tlrl-nacga23) at 10 μg/mL (for stimulation of STING).

Techniques: Cytometry, Expressing, Standard Deviation

Journal: Frontiers in Immunology

Article Title: Tumor-dependent myeloid and lymphoid cell recruitment in genO-BRGSF-HIS mice: a novel tool for evaluating immunotherapies

doi: 10.3389/fimmu.2025.1624724

Figure Lengend Snippet: NK cells are recruited to the A549 TME and display activation markers. (A) Tumor growth was measured over time in genO-BRGSF-HIS mice engrafted with hCD34 + cells from three donors and implanted with 5 x 10 6 A549 cells. (B) Quantification of mouse body weight represented per hCD34 + HSC donor. For graphs (A, B) , seven mice were used for donors 1 and 2, and four mice for donor 3 (18 mice in total). (C) Quantification of the percentage of murine or human CD45 + cells as part of the immune cell infiltrate in A549 tumors at an average size of 200, 400 or 800 mm 3 . Mean percentages are plotted. (D) Quantification of the cell populations present in the human immune infiltrate of the A549 TME at an average size of 200, 500 or 800 mm 3 by cytometry. Mean percentages are plotted. For (C, D) , the graphs represent tumors in six genO-BRGSF-HIS mice per timepoint, engrafted with hCD34 + cells from three donors and implanted with 5 x 10 6 tumor cells. In the 200 mm 3 timepoint, 3 mice were used for donor 1, 2 mice for donor 2 and 1 mouse for donor 3. In the 500 mm 3 timepoint, 2 mice/donor were used. In the 800 mm 3 timepoint, 2 mice were used for donor 1, 3 mice for donor 2 and 1 mouse for donor 3. (E) Quantification of the percentage of different NK cells expressing activation markers, in 200, 400 or 800 mm 3 tumors. nd, not detected. Individual points represent individual mice and the different colors represent different donors (three in total). Error bars represent standard error of the mean. Data were analyzed with a two-way ANOVA. ***P<0.001.

Article Snippet: For splenocyte stimulation, mouse CD45 + cells were depleted and hCD45 + -enriched cells were then stimulated with either CpG ODN 2216 (Invivogen #tlrl-2216) at 10 μM (for stimulation of TLR9); Poly(I:C) (Invivogen # tlrl-picw) at 10 μg/mL (for stimulation of TLR3), R837 (Invivogen # tlrl-imqs) at 10 μg/mL (for stimulation of TLR7); R848 (Resiquimod, Invivogen, #tlrl-r848) at 10 μg/mL (for stimulation of TLR7/8) or 2’3’ cGAMP (Invivogen # tlrl-nacga23) at 10 μg/mL (for stimulation of STING).

Techniques: Activation Assay, Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: Tumor-dependent myeloid and lymphoid cell recruitment in genO-BRGSF-HIS mice: a novel tool for evaluating immunotherapies

doi: 10.3389/fimmu.2025.1624724

Figure Lengend Snippet: HPAF-II tumors in genO-BRGSF-HIS mice have a T-cell dominated TME and respond to T-cell engager combotherapy. (A) Immune cell infiltration was measured in the TME of HPAF-II tumors, and the percentage of murine or human CD45 + cells was calculated relative to the total cells of the tumors. (B) Quantification of the types of cells present in the human immune infiltrate of the HPAF-II TME by cytometry. (C) Quantification of the T cell subpopulations in TME of HPAF-II tumors by cytometry. The graphs in (A–C) represent tumors in 25 hFlt3L-treated genO-BRGSF-HIS mice implanted with 1.5 x 10 6 HPAF-II cells. Individual points represent individual mice. (D) Scheme illustrating the experimental setup for tumor implantation and treatment with bispecific antibodies. (E) Tumor volume was followed over time in genO-BRGSF-HIS mice treated in monotherapy with the NILK-2301 bispecific antibody targeting CD3 and CEACAM5, or in combotherapy using NILK-2301 and NI-3301, which targets CD28 and CEACAM5. The graph represents 12 genO-BRGSF-HIS mice, engrafted with hCD34 + cells from four donors (three mice/donor). Statistical analysis performed on day 29 post engraftment between vehicle and NILK-2301 + NI-3301. Data were analyzed using a Mann Whitney test. i.v.- intravenous injection; i.p.- intraperitoneal injection. (F) Survival curves of genO-BRGSF-HIS mice treated with bispecific antibodies in mono- or combotherapy. Data were analyzed with a Log-rank (Mantel-Cox) test. (G) Individual tumor growth graphs represented by each treatment condition, displaying the tumor growth curves for each group of mice based on the hCD34 + HSC donor. Error bars represent standard deviation. *P<0.05, ***P<0.001.

Article Snippet: For splenocyte stimulation, mouse CD45 + cells were depleted and hCD45 + -enriched cells were then stimulated with either CpG ODN 2216 (Invivogen #tlrl-2216) at 10 μM (for stimulation of TLR9); Poly(I:C) (Invivogen # tlrl-picw) at 10 μg/mL (for stimulation of TLR3), R837 (Invivogen # tlrl-imqs) at 10 μg/mL (for stimulation of TLR7); R848 (Resiquimod, Invivogen, #tlrl-r848) at 10 μg/mL (for stimulation of TLR7/8) or 2’3’ cGAMP (Invivogen # tlrl-nacga23) at 10 μg/mL (for stimulation of STING).

Techniques: Cytometry, Tumor Implantation, MANN-WHITNEY, Injection, Standard Deviation

Journal: bioRxiv

Article Title: Profiling histone post-translational modifications to identify signatures of epigenetic drug response in T-cell acute lymphoblastic leukemia

doi: 10.1101/2025.09.01.673463

Figure Lengend Snippet: (A) NSG mice were engrafted with malignant primary cells derived from the bone marrow of 10 T-ALL patients for tumor expansion. Upon disease establishment, mice were sacrificed, and leukemic cells were harvested from the spleen. To mimic biological replicates, six mice were injected per patient. The percentage of human CD45+ cells (hCD45%) was measured by flow cytometry, with only samples exceeding 85% used for downstream analysis. Global hPTM profiling and DNA methylation sequencing were performed for all 10 PDX models. In parallel, PDX cells were treated ex vivo with a panel of eight epidrugs to assess drug sensitivity. Spearman correlation analysis was then conducted to explore the relationship between hPTM levels and drug response. Figure created with Biorender.com . (B) Clustered and annotated heatmap displaying the normalized and summarized single hPTM levels across the 10 PDX models. (C) Volcano plot illustrating the differential analysis of hPTMs between CIMPlow and CIMPhigh PDX models.

Article Snippet: Lymphoblast engraftment and leukemic burden was monitored by flow cytometric analysis on peripheral blood, using human CD45+ (hCD45) staining (CD45-FITC antibody; Miltenyi Biotec, Bergish Gladbag, Germany).

Techniques: Derivative Assay, Injection, Flow Cytometry, DNA Methylation Assay, Sequencing, Ex Vivo