Journal: Blood Advances

Article Title: CAR T cells targeting CCR4 selectively deplete human Tregs ex vivo and in vivo

doi: 10.1182/bloodadvances.2025017573

Figure Lengend Snippet: Tregs are depleted by the CCR4-CAR in a humanized mouse model. (A) Experimental design. NSG-SGM3-IL15 engrafted with CD34 + hematopoietic stem cells were injected with 1 million CAR + CCR4-CARTs IV. Blood was collected on days 0, 3, 5, and 8, and mice were euthanized on day 11. (B) Representative flow plots showing the proportion of human CD45 (hCD45) and mCD45 leukocytes at baseline. (C) Proportion of Tregs, CD4 + non-Treg, and CD4 − cells of hCD45 percent at baseline. (D) Percentage of Tregs, non-Treg, and CD4 − cells that are CCR4 + at baseline. (E) Representative flow plots showing the CCR4 + and FOXP3 + expression on the CD4 + population before and after CART administration gated on CD4 + cells. (F-K) Proportions of Tregs, CD4 + non-Tregs, and CD4 − cells over time. Significance was determined using t tests corrected for multiple comparisons, with comparison to baseline indicated on graph; ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001. M1, Mouse 1; M2, Mouse 2; M3, Mouse 3; mCD45, mouse CD45.

Article Snippet: Mice were engrafted with human cord blood–derived CD34 + hematopoietic stem cells, and females were available for use at age 13 to 18 weeks after evaluation of engrafted human cell populations by flow cytometry at The Jackson Laboratory.

Techniques: Injection, Expressing, Comparison

Journal: Nature Communications

Article Title: ATRX loss couples genome instability at a G-rich repeat to dysregulation of human alpha-globin expression

doi: 10.1038/s41467-026-69169-7

Figure Lengend Snippet: a Schematic representation of human α-globin locus on chromosome 16p13.3. From the telomeric end, the locus contains the ζ-globin gene HBZ , the minor μ-globin gene HBM , the two α-globin genes HBA2 and HBA1 , and lastly the minor θ-globin gene HBQ1 . Two pseudogenes are present: ψζ HBZP1 between HBZ and HBM , and ψα1 HBAP1 between HBM and HBA2 . Four distal enhancers (R1-R4) are situated upstream of the genes. b Schematic overview of the experimental workflow to generate ATRX knockout erythroid cells from healthy donor CD34 + HSPCs, followed by downstream analysis. c Western blot analysis of ATRX expression in day 7 differentiated cells, confirming efficient knockout. Quantification is presented on the right (n = 3 biological replicates, data shown as mean ± SEM). ** p = 0.009, two-tailed unpaired Welch’s t-test. d Relative expression of HBA, HBM and α to β-like globin ratios by RT-qPCR analysis in day 10 and day 13 differentiated erythroid cells (n = 3 biological replicates, data shown as mean ± SEM). Differences between AAVS1 and ATRX KO were compared using a two-tailed unpaired Welch’s t-test on log₂-transformed fold-change values. e Genotyping summary of the collected 351 BFU-E colonies derived from ATRX KO samples showing genotype distribution. f Three-dimensional plot showing Biomark gene expression analysis of ATRX, HBA and HBM in single BFU-E colony. Each dot represents one colony, and the edited AAVS1 colonies are shown in blue; ATRX KO colonies with frameshift mutations (ATRX_FS) are shown in red. AAVS1 controls gather around the internal space whereas the majority of the ATRX KO colonies assemble around the origin indicating low expression of ATRX, HBM and HBA . g Quantified gene expression analyses of BFU-E colonies show statistical differences between AAVS1 (n = 15) and ATRX KO groups (n = 57) (Mann-Whitney U test). Individual colonies are selected from two biological repeats. *** p = 1.493 × 10 -5 for ATRX expression, ** p = 0.003632 for HBM expression, and p = 0.9083 for HBA expression. ns: not significant. Source data are provided as a Source Data file.

Article Snippet: 1-3 × 10 5 of CD34 + cells were transfected with RNPs (3.2 μg sgRNA (synthesised by Synthego) + 6 μg SpCas9 nuclease (IDT)) using Amaza P3 Primary cell 4D-Nucleofector X Kit (Lonza, Cat No V4XP-3032) and 4D-Nucleofector X Unit device (Lonza).

Techniques: Knock-Out, Western Blot, Expressing, Two Tailed Test, Quantitative RT-PCR, Transformation Assay, Derivative Assay, Gene Expression, MANN-WHITNEY

Journal: Nature Communications

Article Title: ATRX loss couples genome instability at a G-rich repeat to dysregulation of human alpha-globin expression

doi: 10.1038/s41467-026-69169-7

Figure Lengend Snippet: a Schematic representation of human α-globin locus on chromosome 16p13.3. From the telomeric end, the locus contains the ζ-globin gene HBZ , the minor μ-globin gene HBM , the two α-globin genes HBA2 and HBA1 , and lastly the minor θ-globin gene HBQ1 . Two pseudogenes are present: ψζ HBZP1 between HBZ and HBM , and ψα1 HBAP1 between HBM and HBA2 . Four distal enhancers (R1-R4) are situated upstream of the genes. b Schematic overview of the experimental workflow to generate ATRX knockout erythroid cells from healthy donor CD34 + HSPCs, followed by downstream analysis. c Western blot analysis of ATRX expression in day 7 differentiated cells, confirming efficient knockout. Quantification is presented on the right (n = 3 biological replicates, data shown as mean ± SEM). ** p = 0.009, two-tailed unpaired Welch’s t-test. d Relative expression of HBA, HBM and α to β-like globin ratios by RT-qPCR analysis in day 10 and day 13 differentiated erythroid cells (n = 3 biological replicates, data shown as mean ± SEM). Differences between AAVS1 and ATRX KO were compared using a two-tailed unpaired Welch’s t-test on log₂-transformed fold-change values. e Genotyping summary of the collected 351 BFU-E colonies derived from ATRX KO samples showing genotype distribution. f Three-dimensional plot showing Biomark gene expression analysis of ATRX, HBA and HBM in single BFU-E colony. Each dot represents one colony, and the edited AAVS1 colonies are shown in blue; ATRX KO colonies with frameshift mutations (ATRX_FS) are shown in red. AAVS1 controls gather around the internal space whereas the majority of the ATRX KO colonies assemble around the origin indicating low expression of ATRX, HBM and HBA . g Quantified gene expression analyses of BFU-E colonies show statistical differences between AAVS1 (n = 15) and ATRX KO groups (n = 57) (Mann-Whitney U test). Individual colonies are selected from two biological repeats. *** p = 1.493 × 10 -5 for ATRX expression, ** p = 0.003632 for HBM expression, and p = 0.9083 for HBA expression. ns: not significant. Source data are provided as a Source Data file.

Article Snippet: 2 × 10 6 of ATRX degron-VNTR KO differentiated day 6 cells were transfected with RNPs using the 2B Amaxa Human CD34 Cell Nucleofector Kit on the AMAXA Nucleofector 2B system.

Techniques: Knock-Out, Western Blot, Expressing, Two Tailed Test, Quantitative RT-PCR, Transformation Assay, Derivative Assay, Gene Expression, MANN-WHITNEY