Journal: FEBS Open Bio

Article Title: In vitro generation of regulatory T cells: A challenging tool for studying immune aging in humans

doi: 10.1002/2211-5463.70349

Figure Lengend Snippet: (A) Representative dot plots of isolated peripheral blood mononuclear cells (PBMCs) before (upper row) and after (lower row) total CD4 + cells magnetic‐activated cell sorting (MACS) from a young donor. Cells are gated on the living population and checked for CD4 vs. CD8 (left panels), CD45RA vs. CD28 expression (middle panels), and for CD127 vs. CD25 (right panels) expression. Frequencies of CD4 + Foxp3 + (B) and CD25 + of CD4 + Foxp3 + (C) cells are shown after 4 days of in vitro induction of Treg cells in the presence of 10% fetal calf serum (FCS) containing medium RPMI or in serum‐free medium XVivo‐15 (MEAN; n = 2, young donors).

Article Snippet: For the purification of total CD4 + T cells, we used the Miltenyi Biotec CD4 T‐cell Kit (130‐096‐533) according to the manufacturer's protocol.

Techniques: Isolation, FACS, Expressing, In Vitro

Journal: FEBS Open Bio

Article Title: In vitro generation of regulatory T cells: A challenging tool for studying immune aging in humans

doi: 10.1002/2211-5463.70349

Figure Lengend Snippet: Frequency of Foxp3 + CD4 + cells (A) and CD25 expression in Foxp3 + cells (B) after 6 days of induction in TCR‐stimulated cells in the presence of IL‐2 (iTreg IL‐2 ) only or IL‐2 + TGF‐β (iTreg TGFβ ) in young and older individuals. (Mean ± SEM; n ≥ 3). Statistical significance P < 0.05*, and P < 0.01** (two‐way ANOVA with Sidak's post hoc test).

Article Snippet: For the purification of total CD4 + T cells, we used the Miltenyi Biotec CD4 T‐cell Kit (130‐096‐533) according to the manufacturer's protocol.

Techniques: Expressing

Journal: Nature Genetics

Article Title: Single-allele nanoscale mapping of regulatory variants

doi: 10.1038/s41588-026-02776-8

Figure Lengend Snippet: a , Uniform Manifold Approximation and Projection (UMAP) of scATAC–seq from peripheral blood, colored by enrichment of lead SNPs in open chromatin peaks per cluster . b , Clustered hierarchical heatmap for haplotypes with at least one molecular mechanism assigned. Mechanistic assignment weight = 1/(number of distinct candidate mechanisms among SNPs in the haplotype). c , Micro-C from CD4 + T cells (3-kb bin size; pooled from three replicates), ATAC–seq (CD4 + , unstimulated blue, stimulated red, overlay) and CTCF ChIP–seq for the 1q24.3–25.1 locus: MCC for rs75084219 (eczema), rs78037933 (vitiligo), rs1474379684 (celiac disease) and rs62803660 (Crohn’s disease) with SNP–promoter contacts highlighted in gray (3 biological replicates, each with 6 technical replicates (18 in total)). d , Comparison of the number of variant–promoter ligation junctions identified by MCC (median = 828.2) and Micro-C (median = 2.07) ( n = 349 promoter contacts measured across 2 independent libraries for both methods, median (center line), 25th and 75th percentiles (box limits) and whiskers extending to the most extreme values within 1.5× the interquartile range). e , Gene ontology overrepresentation analysis of MCC-prioritized genes in CD4 + T cells (top ten pathways by gene ratio are shown (total significant hits = 251)). The gene ratio is defined as the number of genes in the input list that are annotated to the corresponding term (function) over the number of genes in the input list. f , Comparison between the enrichment of genes with the highest number of MCC contacts (primary nominated gene) versus genes with the second highest number of MCC contacts (secondary nominated gene) compared to background (all genes ±1 Mb from captured variants). For reference, the enrichment scores of the nearest and second nearest genes for each variant are also shown (essentiality (Depmap), drug target and approved drugs (open targets) and primary immunodeficiency (Genomics England)) ( χ 2 test, two-sided P values reported in ). cDCs, conventional dendritic cells; MAIT, mucosal-associated invariant T cells; NK, natural killer cells; pDCs, plasmacytoid dendritic cells; T CM , central memory T cells; T EM , effector memory T cells; T reg , regulatory T cells; B int, intermediate B cells.

Article Snippet: The peripheral blood mononuclear cell layer was isolated and CD4 + or CD3 + pan-T cells were enriched with CD4 + magnetic beads (Miltenyi, cat. no. 130-045-101) or pan-T cell isolation kit (Miltenyi, cat. no. 130-096-535), respectively, as per the manufacturer’s instructions.

Techniques: ChIP-sequencing, Comparison, Variant Assay, Ligation, Analysis

Journal: Nature Genetics

Article Title: Single-allele nanoscale mapping of regulatory variants

doi: 10.1038/s41588-026-02776-8

Figure Lengend Snippet: a , Uniform Manifold Approximation and Projection (UMAP) of single-cell ATAC sequencing from peripheral blood. b , Upset plot of SNP prioritisation pipeline output. A total of 3238 IMID-GWAS signals were analysed, with 1441 having at least 1 SNP being identified in the six molecular mechanisms tested (coding, splicing, enhancer, CTCF, promoter, UTR). A total of 727 were classified as CTCF altering or enhancer altering in activated CD4 + /CD8 + T cells or Monocytes. c , Enrichment scores of IMID-associated SNPs for each molecular mechanism tested in the SNP prioritisation pipeline (n = 124,000) compared to dbSNP151 common variants across the genome (n = 15,000,000). Enrichment scores were calculated as: the fraction of GWAS variants that can be explained by the tested molecular mechanisms over fraction of total common variants explained by the tested molecular mechanism (Χ 2 test, two-sided, 3’UTR ( P = 0.001), coding ( P = 0.145), CTCF ( P = 0.203), promoter ( P = 3.68 × 10 −10 ), splicing (P = 7.97 × 10 −13 , enhancer (4.67 × 10 −53 )). d , 3 kb binned Micro-C, ATAC (CD4 + , unstimulated blue, stimulated red, overlay) and CTCF ChIP-seq for the 10q21.3 locus with MCC from rs12766391 . e , 1.5 kb binned Micro-C, ATAC (CD4 + , unstimulated blue, stimulated red, overlay) and CTCF ChIP-seq for the RUNX3 locus with MCC from rs57974094 (green) and rs4265380 (blue). cDCs, conventional dendritic cells; MAIT, mucosal-associated invariant T cells; NK, natural killer cells; pDCs, plasmacytoid dendritic cells; T cm , central memory T cells; T em , effector memory T cells; T reg , regulatory T cells; B int, intermediate B cells.

Article Snippet: The peripheral blood mononuclear cell layer was isolated and CD4 + or CD3 + pan-T cells were enriched with CD4 + magnetic beads (Miltenyi, cat. no. 130-045-101) or pan-T cell isolation kit (Miltenyi, cat. no. 130-096-535), respectively, as per the manufacturer’s instructions.

Techniques: Single Cell, Sequencing, ChIP-sequencing

Journal: Nature Genetics

Article Title: Single-allele nanoscale mapping of regulatory variants

doi: 10.1038/s41588-026-02776-8

Figure Lengend Snippet: a , Count data for PCR deduplicated reads containing the target SNP for a subset (donor 1 only) of heterozygous SNPs (n = 147 variant read counts from 6 independent MCC libraries from a single donor). This data is used to variant call the genotype of captured GWAS sites before applying the MCCv single-allele analysis pipeline. b , Summary of the number of variants captured by MCC (total) and those amenable to allelic analysis (heterozygous). c , Visualisation of the total number of heterozygous variants and the breakdown of those where allelic data was successfully generated using MCCv. We also include a theoretical estimation of the number of variants that could generate allele-specific data in a dpnII-based 3 C experiment (for example Capture C), where variants had to fall within 60 bp of a dpnII-cut site to be classified as successful. d , The distribution of SNPs in the MCCv dataset in terms of percentage of peaks which are significantly altered between alleles (relative to the total number of MCCv peak for that SNP). e , as in Fig. but data has been filtered for promoters, CTCF, or enhancer elements before plotting. f , MCCv profile for rs9526002 , with SNP-promoter contact highlighted in grey. Difference plots show alternate-minus-reference contacts, with CD4 + ATAC-seq and CTCF ChIP-seq tracks below. g , rs10950648 nanoscale contact maps generated using 10-bp directional windowing and mirrored on the diagonal. The AP-1 motif and enhancer sequence are shown, with the effect of the alternate allele highlighted (n = 12 (2 donors, 6 independent MCC libraries each), merged β = −1.10).

Article Snippet: The peripheral blood mononuclear cell layer was isolated and CD4 + or CD3 + pan-T cells were enriched with CD4 + magnetic beads (Miltenyi, cat. no. 130-045-101) or pan-T cell isolation kit (Miltenyi, cat. no. 130-096-535), respectively, as per the manufacturer’s instructions.

Techniques: Variant Assay, Analysis, Generated, Capture-C, ChIP-sequencing, Sequencing

Journal: Nature Genetics

Article Title: Single-allele nanoscale mapping of regulatory variants

doi: 10.1038/s41588-026-02776-8

Figure Lengend Snippet: a , Alternate or reference normalized junction counts at MCCv peaks for heterozygous SNPs ( n depends on how many donors are heterozygous for the SNP from which the peak is derived (minimum of six independent libraries per donor), paired, two-sided Student’s t -test, local false discovery rate (FDR) estimation). b , Clustered hierarchical heatmap of the average ratio of alternate to reference junction counts for peaks overlapping different regulatory element categories per SNP. c , Percentage of variants with significant MCCv changes (two-sided χ 2 test, disease variants n = 227). Enrichment over controls (enrichment, P , n control): cell type = IMID variants intersecting monocyte enhancers (7.5, 0.0015 and 14); haplotype = SNPs in haplotype that do not intersect CD4 + T cell enhancers (7.5, 2.73 × 10 −13 and 82); enhancer = variants that intersect CD4 + T cell enhancers but are not associated with IMIDs (2.2, 0.000014 and 76). d , MCCv from rs10950648 with SNP–promoter contact in gray. Difference plots (alternate minus reference), ATAC–seq (CD4 + , unstimulated blue, stimulated red) and CTCF ChIP–seq shown underneath. e , Nanoscale contact maps of rs10950648 with 10-bp directional windowing. They are mirrored on the diagonal. PRDM1 motif and enhancer sequences are shown with the effect of the alternate allele highlighted ( n = 12, merged β = -1.09). f , ATAC–seq read skew for rs10950648 ( q = 0.024) and rs9526002 ( q = 0.025) ( n = 2 donors, mean ± s.d.). g , Allelic expression of primary or secondary nominated genes phased to a heterozygous variant: 88 variants (102 variant–gene pairs) tested. Twenty-three genes showed donor-level skew (variable-phased SNPs ( n ) per gene; two-sided, Wilcoxon’s signed-rank test was used per donor (Fisher’s method across donors) with the Benjamini–Hochberg correction). h , Percentage of allelically imbalanced genes by SNP–gene categories. Backgrounds were genome-wide allelic gene imbalance, 10.0% (231 out of 2,313) and allelic gene imbalance without MCCv skew, 8.9% (17 out of 190). The percentage of genes showing allelic imbalance linked to variants with evidence of MCCv changes (category: n variant-–gene pairs, enrichment over genome-wide ( p ), enrichment over MCCv ( p ); secondary genes: 6 of 23, 2.61 (0.0316); primary genes 10 of 33, 3.03 (0.0316), 3.40 (0.038); primary or secondary: 16 of 45, 3.55 (0.0001), 3.99 (0.0002) (two-tailed χ 2 test).

Article Snippet: The peripheral blood mononuclear cell layer was isolated and CD4 + or CD3 + pan-T cells were enriched with CD4 + magnetic beads (Miltenyi, cat. no. 130-045-101) or pan-T cell isolation kit (Miltenyi, cat. no. 130-096-535), respectively, as per the manufacturer’s instructions.

Techniques: Derivative Assay, Control, ChIP-sequencing, Expressing, Variant Assay, Genome Wide, Two Tailed Test

Journal: Nature Genetics

Article Title: Single-allele nanoscale mapping of regulatory variants

doi: 10.1038/s41588-026-02776-8

Figure Lengend Snippet: a , Schematic of converting nanoscale heatmaps into directionally windowed heatmaps for the visualisation of contacts between transcription factor motifs. Reads that give rise to ligation junctions are thought to be protected from MNase cutting by bound transcription factors. The direction of the extension of the reads can be leveraged to estimate the position of the putatively bound transcription factor. This converts the negative signal of the transcription factor footprint into positive signal equating to potential contacts between transcription factors. b , MCC ultra tile over the rs10950648 -containing enhancer (n = 6 independent MCC libraries, bin size = 2, tile = 2.2 kb). Corresponding ATAC (CD4 + , unstimulated blue, stimulated red, overlay) and a unified CTCF track are shown underneath. The capture probe/SNP locations are shown by the horizontal bar and red triangle respectively. Zoom in shows MCCv single-allele directional windowed heatmaps for reference and alternate alleles at the variant shown in Fig. . c , As in b for the rs9526002 -containing enhancer with zoom in corresponding to directional windowed heatmap as shown in Extended Data Fig. (n = 6 independent MCC libraries, bin size = 2, tile = 1.9 kb). d and e , Footprinting data from ATAC-seq combined with MCCv based capture enrichment (above) and 1D-MCCv footprinting (below) for both reference and alternate alleles of rs10950648 and rs9526002 . SNP position is indicated with the red triangle.

Article Snippet: The peripheral blood mononuclear cell layer was isolated and CD4 + or CD3 + pan-T cells were enriched with CD4 + magnetic beads (Miltenyi, cat. no. 130-045-101) or pan-T cell isolation kit (Miltenyi, cat. no. 130-096-535), respectively, as per the manufacturer’s instructions.

Techniques: Ligation, Variant Assay, Footprinting

Journal: Nature Genetics

Article Title: Single-allele nanoscale mapping of regulatory variants

doi: 10.1038/s41588-026-02776-8

Figure Lengend Snippet: a , Effect size estimate of GLM from basepairC at tested SNPs (n = 231) (bottom 20% of variants based on read counts were filtered). Estimates were done between maps of different genotypes (allele-specific) (Genotype, mean β-coefficient (effect size) = 0.293). Random combinations of 6 reference and 6 alternate allele sample matrices were compared (mean β-coefficient = 0.097) (median (centre line), 25th and 75th percentiles (box limits), and whiskers extending to most extreme values within 1.5 × interquartile range). b , Effect size from basepairC matrix difference modelling in Jurkat controls (prioritised using ATAC skew) vs. T cell disease associated enhancers (prioritised by ATAC intersection only). Variants within lowest 10% of read counts for MCCv nanoscale maps were filtered from the correlation. Gain-of-function (MCCv nanoscale effect size ≥ 0.2) and loss-of-function (MCCv nanoscale effect size ≤ -0.2) variants are highlighted in green and purple respectively. Variants highlighted throughout the study are indicated on the plot (Jurkat controls – n = 38 variants (6 independent MCC libraries), IMID variants – n = 191 variants (6 independent MCC libraries from 1-3 biological donors, median (centre line), 25th and 75th percentiles (box limits), and whiskers extending to the most extreme values within 1.5 × the interquartile range.). c , Allelic ratio from ATAC-seq versus effect size from basepairC matrix difference modelling (n = 201, two-sided Spearman’s ρ = 0.62, P = 2.2 × 10 −16 ). Variants within the lowest 10% of read counts for either ATAC or MCCv were filtered from the correlation. Nominally gain-of-function (ATAC allelic ratio ≥ 0.55 & MCCv nanoscale effect size ≥ 0.05) and loss-of-function (ATAC allelic ratio ≤ 0.45 & MCCv nanoscale effect size ≤ -0.05) variants are highlighted in green and purple respectively.

Article Snippet: The peripheral blood mononuclear cell layer was isolated and CD4 + or CD3 + pan-T cells were enriched with CD4 + magnetic beads (Miltenyi, cat. no. 130-045-101) or pan-T cell isolation kit (Miltenyi, cat. no. 130-096-535), respectively, as per the manufacturer’s instructions.

Techniques:

Journal: Nature Genetics

Article Title: Single-allele nanoscale mapping of regulatory variants

doi: 10.1038/s41588-026-02776-8

Figure Lengend Snippet: a , Associations of rs4409785 from the National Center for Biotechnology Information (NCBI) GWAS catalog, with −log 10 ( P ) versus log 10 (odds ratio) for the tested allele plotted as a volcano. Associations reported with reference T allele are shown in purple, whereas associations reported with the alternate C allele are shown in green. Representative autoimmune disease signals are labeled (autoimmune thyroid disease (ATD), RA, MS, myasthenia gravis (MG) and vitiligo). b , CTCF consensus motif and the sequence around rs4409785 in humans. The effect of rs4409785 is highlighted in gray. Sequence alignment from early hominins and chimps is shown below. c , MCCv basepair resolution footprinting for reference and alternate alleles with the rs4409785 location marked by a red triangle. Corresponding nanoscale heatmaps are shown as in Fig. (bin size = 1 bp, 800-bp window, n = 6 merged per allele and merged β = 1.09). d , CTCF ChIP–seq ( q = 0.000123), ATAC–seq ( q = 0.000123) and H3K27ac ChIP–seq ( q = 0.156) skew in heterozygous donors ( n = 15 CTCF or ATAC, n = 12 H3K27ac; two-sided Wilcoxon’s matched-pairs, signed-rank test, two-stage step-up procedure for multiple-test correction, mean ± s.d.). e , CTCF ChIP–seq and ATAC–seq reads per kilobase of transcript per million mapped reads-normalized signal from three donors who are either homozygous reference (purple) or alternate (green) for rs4409785 . f , MCCv profiles for rs4409785 alleles with corresponding ATAC (CD4 + , unstimulated blue, stimulated red, overlay) and a unified CTCF track. Capture probe location or SNP is marked by the red triangle and CTCF elements with orientation are marked by purple triangles.

Article Snippet: The peripheral blood mononuclear cell layer was isolated and CD4 + or CD3 + pan-T cells were enriched with CD4 + magnetic beads (Miltenyi, cat. no. 130-045-101) or pan-T cell isolation kit (Miltenyi, cat. no. 130-096-535), respectively, as per the manufacturer’s instructions.

Techniques: Labeling, Sequencing, Footprinting, ChIP-sequencing

Journal: Nature Genetics

Article Title: Single-allele nanoscale mapping of regulatory variants

doi: 10.1038/s41588-026-02776-8

Figure Lengend Snippet: a , Schematic illustrating coupling of basepair-level editing technologies with basepair resolution MCCv. Incomplete genome editing is leveraged to make a population of cells with mixed alleles become deconvoluted with the platform’s single-allele capabilities. b , Base editing experimental design with CTCF guide RNA sequence as well as locations of rs4409785 and the CTCF motif shown. The location of an upstream control guide outside the extended CTCF motif but within the MCC capture oligonucleotide is also shown. c , Experimental set-up of the CTCF and control edits in primary T cells. Edits were performed in parallel and mixed to avoid double editing. The cell mix was then fixed and captured by MCCv. As these cells were heterozygous for risk variant rs4409785 this led to six different editing outcomes that were deconvoluted by single-allele MCCv. To increase the depth of the data, six independent replicates were performed. d , Allele-specific (relative to rs4409785 ) editing efficiency of BE4max in primary CD4 + T cell for both control and CTCF guides. Editing efficiencies are shown as a percentage of NGS reads with C-to-T substitution. Where multiple Cs could be edited, the one with highest editing efficiency is shown ( n = 2 independent experiments, mean ± s.d.). e , Edit-MCCv tracks for reference (purple) and alternate (green) alleles of rs4409785 with wild-type, control edit and CTCF edit shown. A CTCF ChIP–seq track is shown underneath. f , Nanoscale heatmaps for rs4409785 reference and alternate alleles with corresponding edit type (bin size = 1 bp, 800-bp window, n = 6 merged per allele). NGS, next-generation sequencing.

Article Snippet: The peripheral blood mononuclear cell layer was isolated and CD4 + or CD3 + pan-T cells were enriched with CD4 + magnetic beads (Miltenyi, cat. no. 130-045-101) or pan-T cell isolation kit (Miltenyi, cat. no. 130-096-535), respectively, as per the manufacturer’s instructions.

Techniques: Sequencing, Control, Variant Assay, ChIP-sequencing, Next-Generation Sequencing

Journal: Nature Genetics

Article Title: Single-allele nanoscale mapping of regulatory variants

doi: 10.1038/s41588-026-02776-8

Figure Lengend Snippet: a , Ligation junction counts between rs4409785 and the major downstream CTCF site (wild type vs. edit; q = 0.089 (reference) and q = 0.000001 (alternate), wild type vs. control; q = 0.141 (reference) and q = 0.079 (alternate)) (n = 6 independent MCC libraries across 1 biological donor with 3 independent nucleofections, Student’s t-test, two-sided, two-stage step-up procedure for multiple test correction, mean ± s.d.). b , Detailed schematic for MCCv phasing strategy employed in order to analyse super-enhancer–promoter contacts and RNA in an allele-specific manner. Zoom-in shows the locations of phased intronic SNPs for both SESN3 and FAM76B used in RNA skew analysis. c , Rank Ordering of Super-enhancers (ROSE) analysis of H3K27ac ChIP-seq data from activated CD4 + T cells. The dashed line marks the inflection point above which enhancers are classified as super-enhancers (SE). The SESN3 super-enhancer is labelled in red and is ranked as the 75 th strongest super-enhancer out of 760 super-enhancers and 15,281 total enhancers. d , RPKM normalised ATAC signal at a genomic window corresponding to the defined super enhancer in a variety of blood cell types with both unstimulated (blue) and stimulated (green) paired data.

Article Snippet: The peripheral blood mononuclear cell layer was isolated and CD4 + or CD3 + pan-T cells were enriched with CD4 + magnetic beads (Miltenyi, cat. no. 130-045-101) or pan-T cell isolation kit (Miltenyi, cat. no. 130-096-535), respectively, as per the manufacturer’s instructions.

Techniques: Ligation, Control, Analysis, ChIP-sequencing

Journal: Nature Genetics

Article Title: Single-allele nanoscale mapping of regulatory variants

doi: 10.1038/s41588-026-02776-8

Figure Lengend Snippet: a , A 2.5-kb, binned Micro-C, ATAC–seq (CD4 + , unstimulated blue, stimulated red, overlay) and CTCF ChIP–seq for the 11q21 locus. Bulk MCCs from the promoters of SESN3 and FAM76B are shown in blue. The location of the super-enhancer is highlighted in gray. b , Phased enhancer–promoter contacts ( SESN3 ( q = 0.018, n = 24 peaks across six independent MCC libraries) and FAM76B ( q = 0.916, n = 24 peaks across six independent MCC libraries), Student’s t -test, two-stage step-up procedure for multiple-test correction, mean ± s.e.m.) and intron-rich RNA ( SESN3 ( q = 0.00054, n = 37 phased heterozygous variants across two donors) and FAM76B ( q = 0.11, n = 45 phased heterozygous variants across two donors), Wilcoxon’s signed-rank test, two-stage step-up procedure for multiple-test correction, mean ± s.e.m.) from MCCv-phased heterozygous variants for both SESN3 and FAM76B . c , Working mechanistic model for the 11q21 risk locus. The neo-CTCF site competes with canonical upstream boundary elements for convergent downstream CTCF sites, forming a subdomain that isolates the super-enhancer away from the promoter of SESN3 .

Article Snippet: The peripheral blood mononuclear cell layer was isolated and CD4 + or CD3 + pan-T cells were enriched with CD4 + magnetic beads (Miltenyi, cat. no. 130-045-101) or pan-T cell isolation kit (Miltenyi, cat. no. 130-096-535), respectively, as per the manufacturer’s instructions.

Techniques: ChIP-sequencing

Journal: Nature Genetics

Article Title: Single-allele nanoscale mapping of regulatory variants

doi: 10.1038/s41588-026-02776-8

Figure Lengend Snippet: a , Experimental design of the mouse EAE model. b , Clinical EAE score for both Rosa26 and Sesn3 cohorts. For each day where both cohorts were represented, scores were compared statistically ( n = 6 mice per condition, day 12, q = 0.0024; day 13, q = 0.0089; day 14, q = 0.0024; two-sided, unpaired, Student’s t -test with single pooled variance, mean ± s.e.m.). c , Dot plot representation of SESN gene family members’ scRNA-seq expression from synovial tissue of patients wth RA . d , Representative flow cytometry contour plots showing a positive population of CD4 + ( y axis) and phospho-4E-BP1 + (mTORC1 signaling marker, x axis). e , Western blots for FLAG-tagged SESN3 or GATOR2 complex subunit, WDR59, either from cell lysate (input) or from co-immunoprecipitation using anti-FLAG antibody-conjugated beads. Experimental conditions compared were a medium control (fed state) versus a starved state in Hanks’ balanced salt solution (HBSS) with or without various free amino acids. Specific indicated free amino acids were added to the starved state during the 1-h starvation at 500 µM. f , Schematic for working model of the effect of rs4409785 on reducing SESN3 gene expression and, in turn, how this may affect mTORC1 signaling in starvation conditions, specifically low tryptophan availability. AAVS1 , adeno-associated virus integration site 1; CNS, central nervous system; E-P, enhancer–promoter; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IFN, interferon; IP, immunoprecipitation; MOG, myelin oligodendrocyte glycoprotein; MT hi , mitochondrial high; mTORC1, mTOR complex 1; PTX, pertussis toxin.

Article Snippet: The peripheral blood mononuclear cell layer was isolated and CD4 + or CD3 + pan-T cells were enriched with CD4 + magnetic beads (Miltenyi, cat. no. 130-045-101) or pan-T cell isolation kit (Miltenyi, cat. no. 130-096-535), respectively, as per the manufacturer’s instructions.

Techniques: Expressing, Tissue, Flow Cytometry, Marker, Western Blot, Co-Immunoprecipitation Assay, Control, Gene Expression, Virus, Immunoprecipitation

Journal: Nature Genetics

Article Title: Single-allele nanoscale mapping of regulatory variants

doi: 10.1038/s41588-026-02776-8

Figure Lengend Snippet: a , % edited reads, 24 h post-nucleofection in mHSPCs for Rosa26 and Sesn3 guides (n = 1). b , CD45.1 + % in different cell types; CD8 + (q = 0.0029), CD4 + (q = 0.0098), Myeloid (q = 0.344), B cells (q = 0.723) in Rosa26 or Sesn3 conditions (n = 7 mice per condition, Student’s t-test single pooled variance, two-sided, unpaired, two-stage step-up procedure for multiple test correction, mean ± s.d.). c , Relative mouse weights for Rosa26 and Sesn3 cohorts (day 12, q = 0.023; day 13, q = 0.026; day 14, q = 0.026) (n = 6 mice per condition, Student’s t-test single pooled variance, two-sided, unpaired, two-stage step-up procedure for multiple test correction, mean ± s.e.m.). d , Survival curve post EAE-induction for Rosa26 and Sesn3 conditions ( P = 0.019, n = 6, Grehan-Brislow-Wilcoxon test, two-sided). e , % infiltrating activated T cells (CD4 + IFNγ + IL17 + CD44 high ) in CNS for Rosa26 and Sesn3 cohorts ( P = 0.0038, Student’s t-test, two-sided, n = 6 mice per condition, mean ± s.d.). f , Rooted phylogenetic maximum-likelihood tree of SESN gene family from amino-acid sequence. g , Single-cell RNA-seq UMAP from healthy volunteer PBMCs ( SESN3 expression highlighted in green). h , Experimental scheme for ex vivo human T cell editing in fed and starved conditions. i , Tracking of indels by decomposition analysis 72 h post-nucleofection of T cells for AAVS1 and SESN3 sgRNAs (n = 3 nucleofections, mean ± s.d.). j , Phospho-4E-BP + CD4 + T cells % for AAVS1 or SESN3 sgRNA nucleofections in media ( P = 0.462) or starvation ( P = 0.014) (n = 5 nucleofections across donors, Student’s t-test, two-sided, median, interquartile range (box; 25th–75th percentiles), whiskers extending to the most extreme data points within 1.5 × the interquartile range ) . k , Immunoblot for anti-FLAG-SESN3 in wildtype (WT), FLAG knock-in (Tag), and FLAG knock-in edited with sgRNAs targeting SESN3 (Tag-KO).

Article Snippet: The peripheral blood mononuclear cell layer was isolated and CD4 + or CD3 + pan-T cells were enriched with CD4 + magnetic beads (Miltenyi, cat. no. 130-045-101) or pan-T cell isolation kit (Miltenyi, cat. no. 130-096-535), respectively, as per the manufacturer’s instructions.

Techniques: Sequencing, Single Cell, RNA Sequencing, Expressing, Ex Vivo, Analysis, Western Blot, Knock-In

Journal: Frontiers in Immunology

Article Title: A fully human afucosylated anti-GARP antibody PBA-0111 drives anti-tumor immunity through combined ADCC induction and TGF-β1 pathway blockade

doi: 10.3389/fimmu.2026.1921265

Figure Lengend Snippet: PBA-0111 demonstrates potent ADCC against GARP and GARP/TGF-β1 positive tumor cells and Tregs, accompanied by NK cell activation. (A) Raji-GARP target cells were treated with serial dilutions (0.00001-100 nM) of PBA-0111, DS-1055a-like antibody, or afucosylated isotype control and subjected to NK cell-mediated lysis for 4 h at 37 °C at an Effector: Target ratio of 10:1. Cell killing was analyzed by flow cytometry using live/dead stain. Data are shown as mean ± SD from a representative pool of three independent donors tested in duplicate. (B) ADCC was performed against Raji-GARP/TGF-β1 target cells (n=6, from 3 independent donors) using the same conditions as in (A) . (C) ADCC against the L-428 lymphoma cell line (n=6, from 3 independent donors). (D) ADCC against in vitro differentiated Tregs. CD4+CD25high T cells isolated from healthy donor PBMCs were stimulated with anti-CD3/CD28 beads to generate regulatory T cells, then subjected to autologous NK-mediated cellular lysis overnight at 37 °C in the presence of indicated anti-GARP antibodies. Data represent pooled results from three independent donors tested in duplicate, mean ± SD. (E) PBMCs (n=3 donors, in 2 replicates) were co-incubated with Raji-GARP/TGF-β1 cells and either PBA-0111, DS-1055a-like antibody, or afucosylated isotype control (IgG.aF) at 5 nM for 4 hours. NK cells were then analyzed by flow cytometry for CD107a expression in the presence of Golgi stop. (F) The same NK cells were analyzed for IFN-γ and (G) TNF-α production. Data are expressed as fold change relative to the afucosylated isotype control. Statistical significance versus IgG1.aF was assessed by Kruskal-Wallis with Dunn´s multiple comparison, *p< 0.05, **p< 0.01, ***p< 0.001.

Article Snippet: Regulatory T cells were isolated from PBMCs from the same donor as NK cells by immunomagnetic two-step procedure with human CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, 130-091-301) according to the manufacturer’s protocol.

Techniques: Activation Assay, Control, Lysis, Flow Cytometry, Staining, In Vitro, Isolation, Incubation, Expressing, Comparison

Journal: Frontiers in Immunology

Article Title: A fully human afucosylated anti-GARP antibody PBA-0111 drives anti-tumor immunity through combined ADCC induction and TGF-β1 pathway blockade

doi: 10.3389/fimmu.2026.1921265

Figure Lengend Snippet: PBA-0111 activates T cells leading to anti-tumor efficacy. (A–D) CD34 + HSC humanized NSG-hIL15 mice (n=6-7/group) were inoculated subcutaneously with Raji-GARP/TGF-β1 and treated with vehicle or six doses of PBA-0111 or DS-1055a-like antibody at 10 mg/kg twice weekly, initiated when the tumor volumes reached 80-150 mm 3 . At study termination, tumors, spleens and terminal blood were collected and processed for FACS analysis. (A) Proportion of CD8 + cells within the tumor (B) Intratumoral CD8 + /Treg ratio. (C) Activation status of intratumoral CD8 + T cells. (D) NK cell activation in peripheral blood. (E–I) CD34 + HSC humanized NCG-hIL15 mice (n=8/group) were inoculated subcutaneously with HT-29 colon carcinoma cells and treated as above. At study termination, tumors and terminal blood were processed for FACS analysis (n=4-8/group). (E) Proportion of intratumoral CD8 + T cells. (F) Intratumoral CD8+/Treg ratio. (G) CD8 + T cell infiltration confirmed by immunohistochemistry (IHC). (H) Granzyme B expression detected by IHC. (I) Representative IHC images showing CD8 + (green) and Granzyme B + (magenta) cells in immune-positive clusters at the tumor periphery (rim) of HT-29 tumors. Scale bar = 100 µm. Statistical significance was assessed by Kruskal-Wallis test with Dunn´s multiple comparisons. **p < 0.01.

Article Snippet: Regulatory T cells were isolated from PBMCs from the same donor as NK cells by immunomagnetic two-step procedure with human CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, 130-091-301) according to the manufacturer’s protocol.

Techniques: Analysis, Activation Assay, Immunohistochemistry, Expressing

Journal: Frontiers in Immunology

Article Title: A fully human afucosylated anti-GARP antibody PBA-0111 drives anti-tumor immunity through combined ADCC induction and TGF-β1 pathway blockade

doi: 10.3389/fimmu.2026.1921265

Figure Lengend Snippet: PBA-0111 shows signs of T cell activation in soft tissue sarcoma patients following intratumoral administration. (A) CIVO workflow. PBA-0111 was co-formulated with fluorescent microspheres and loaded into the CIVO injector, which delivers eight individual columns of drug directly into the patient tumor (left). STS patients receiving the multiplexed microinjection were evaluated immediately following the procedure by visualization of individual injection sites using a blue light and yellow filter that illuminates the CIVO GLO fluorescent microspheres with an ultraviolet flashlight (center). Following a pre-scheduled surgical resection 2-96 hours later, the tumors were sectioned transverse to the drug columns, yielding 2-4 sections of FFPE tissue for downstream IHC and ISH staining and image analysis. (B) GARP expression assessed by ISH. Representative injection site images from vehicle and PBA-0111-treated sites in Patient 5, with summary data showing the median percentage of GARP + cells across all evaluable injection sites in all patients (n=6). (C) Regulatory T cell infiltration assessed by IHC. Representative injection site images for vehicle and PBA-0111-treated sites in Patient 5, with summary data showing the median percentage of CD4+ T cells that are FoxP3 + across all evaluable injection sites in all patients. (D) . CD8 + cytotoxic T cell activity assessed by IHC. Representative injection site images for vehicle and PBA-0111-treated sites in Patient 5, with summary data showing the median percentage of CD8 + T cells that are Granzyme B + across all evaluable injection sites in all patients.

Article Snippet: Regulatory T cells were isolated from PBMCs from the same donor as NK cells by immunomagnetic two-step procedure with human CD4 + CD25 + Regulatory T Cell Isolation Kit (Miltenyi Biotec, 130-091-301) according to the manufacturer’s protocol.

Techniques: Activation Assay, Tissue, Microinjection, Injection, Staining, Analysis, Expressing, Activity Assay