Journal: Cell Reports Methods

Article Title: Integrating transcription-factor abundance with chromatin accessibility in human erythroid lineage commitment

doi: 10.1016/j.crmeth.2022.100188

Figure Lengend Snippet: GATA-1-high BM progenitors show strong priming for erythroid lineage (A) BM aspirate is ficolled and enriched for CD34 + cells before gating for CD34 + /CD38 + cells and selecting GATA-1-high population (top ∼8%) and remaining GATA-1-expressing cells (denoted as GATA-1 mid/low) (bottom ∼87%). (B) InTAC-seq genome coverage plots at GATA1 and SPI1 loci for GATA-1-high and -mid/low BM progenitors. (C) Heatmap of chromVAR deviation scores across GATA-1 high and mid/low BM progenitors for top 50 most variable motifs. (D) MA plot of log2 fold change in accessibility between GATA-1 high and mid/low BM progenitors versus log2 mean number of reads in consensus peaks. Peaks with significant changes in accessibility are highlighted in red or blue. (E) Most significantly enriched TF motifs in differentially accessible peaks between GATA-1-high and -mid/low BM progenitors calculated using Fisher’s test. (F) (Top) Average accessibility of GATA-1 motif sites across all consensus ATAC-seq peaks binned by GATA-1 motif score. Accessibility is defined here as the area under the curve of a plot of bias-corrected, normalized Tn5 insertions centered at GATA-1 motif sites, integrated from −50 to +50 bp and excluding the TF footprint from −10 to +10 bp. (Bottom) Difference in GATA-1 motif accessibility between GATA-1 high and mid/low samples normalized to the accessibility in the GATA-1 mid/low population for each motif score bin. (G) UMAP of previously published and annotated BM scATAC dataset with Seurat clusters manually annotated as key BM populations. (H) Normalized GATA1 gene expression across BM progenitors in UMAP space (expression derived from scRNA-seq data integrated with scATAC-seq data). (I) Bulk BM progenitor InTAC-seq data simulated as scATAC counts and projected onto scATAC UMAP space.

Article Snippet: Next, BMMNC were either cryopreserved in FBS with 10% of DMSO or previously enriched for CD34 + (CD34 MicroBead Kit, Miltenyi Biotec) before cryopreservation.

Techniques: Expressing, Gene Expression, Derivative Assay

Journal: Cell Reports Methods

Article Title: Integrating transcription-factor abundance with chromatin accessibility in human erythroid lineage commitment

doi: 10.1016/j.crmeth.2022.100188

Figure Lengend Snippet: High-dimensional, single-cell proteomic analysis of BM progenitors identifies TF and surface-marker trends in erythroid commitment (A) BM aspirate is ficolled and enriched for CD34 + cells before staining with antibodies to surface marker panel and key TF to capture single-cell protein abundance using mass cytometry. High-dimensional data with over 1 million cells were used to delineate heterogeneity in BM progenitors and find surface-marker surrogates for GATA-1 TF abundance for further functional validation. (B) Force-directed layout (ForceAtlas2) of density downsampled (to 250,000 cells) CD45 + -gated BM progenitors colored by manually gated populations. (C) Normalized marker expression of key surface and TF proteins (in orange) across force-directed layout. Orange arrow denotes GATA-1-high region in single-cell map. (D) Leiden clusters of BM progenitors (resolution = 1) visualized on force-directed layout. (E) Barplot of frequency of manually gated BM progenitor populations across 11 Leiden clusters. (F) Normalized diffusion pseudotime calculation visualized on force-directed layout with trajectory from HSCs to erythroid-primed progenitors across Leiden clusters 3, 1, and 11 (in red). (G) Row-normalized heatmap of median marker abundance at 100 bins across diffusion pseudotime-aligned trajectory. Orange font and arrows indicate TF protein trends, and bold font with black arrows indicate key surface marker trends in trajectory. (H) Column-normalized heatmap of mutual information scores calculated on cells in Leiden clusters 2, 7, and 8 and normalized across key TF and surface-marker pairs.

Article Snippet: Next, BMMNC were either cryopreserved in FBS with 10% of DMSO or previously enriched for CD34 + (CD34 MicroBead Kit, Miltenyi Biotec) before cryopreservation.

Techniques: Marker, Staining, Quantitative Proteomics, Mass Cytometry, Functional Assay, Biomarker Discovery, Expressing, Diffusion-based Assay

Journal: Cell Reports Methods

Article Title: Integrating transcription-factor abundance with chromatin accessibility in human erythroid lineage commitment

doi: 10.1016/j.crmeth.2022.100188

Figure Lengend Snippet: Surface-marker-defined BM population surrogate for high GATA-1 protein abundance clonally enriches for erythroid lineage (A) Spearman correlation plot of surface markers and GATA-1 abundance in BM progenitors as measured by mass cytometry. (B) Top 8% of GATA-1-expressing cells in CD34 + /CD38 + BM progenitors gated as GATA-1-high BM cells, and remaining GATA-1-mid/low cells used for subsequent analysis. (C) Boxplots of normalized surface-marker abundance of GATA-1-high BM progenitors (top ∼8% of expression) and GATA-1-mid/low BM progenitors (bottom ∼87% of expression) from mass cytometry. (D) Violin plots of GATA-1 protein abundance in manually gated target populations (as defined by CD71 + , CD84 + , CD33 – ), CD123 – MEP populations, and in other BM progenitor populations. (E) Boxplot of clonal differentiation frequency of target population and CD123 - MEP population to different lineages/population types across 4 biological replicates. (p values calculated using Student's t test)

Article Snippet: Next, BMMNC were either cryopreserved in FBS with 10% of DMSO or previously enriched for CD34 + (CD34 MicroBead Kit, Miltenyi Biotec) before cryopreservation.

Techniques: Marker, Quantitative Proteomics, Mass Cytometry, Expressing

Journal: Cell Reports Methods

Article Title: Integrating transcription-factor abundance with chromatin accessibility in human erythroid lineage commitment

doi: 10.1016/j.crmeth.2022.100188

Figure Lengend Snippet: High GATA-1 protein abundance delineates epigenetic program for erythroid commitment in RBC developmental trajectory (A) BM aspirate is ficolled and enriched for CD34 + cells before gating for CD123 – MEP population (CD34 + /CD38 + /CD10 – /CD45RA – /CD123 – ) and selecting high- (25%–40%) and mid- (∼lower 30%) GATA-1-expressing cells within each compartment. (B) GATA-1-high cells from CD34 + /CD38 + and CD123 – MEP compartments InTAC-seq data were simulated as scATAC counts and projected on scATAC UMAP space. (C) Putative erythropoiesis trajectory constructed from HSCs to late erythoid populations and overlaid on scATAC UMAP. (D) Heatmap of top variable TFs by ChromVAR deviation scores across constructed erythroid trajectory with the projected position of GATA-1-high InTAC-seq samples indicated in red as the point of GATA-1-high overlap, in blue as the before point, and in green as the after point. Top: line plot of InTAC-seq-denoted GATA-1-high-simulated scATAC cells as binned across pseudotime. (E) Top 20 genes significantly enriched (of fold change 2 and above) in integrated scRNA-seq data between the 3 bins, before, at, and after GATA-1-high overlap points in trajectory. (F) Summary schematic of continuous differentiation to erythrocytes in BM with downregulation of lymphoid/myeloid TF activity and gene expression programs and upregulation of erythroid TF activity and gene expression programs. High GATA-1 protein abundance overlaps epigenetic program shift to erythroid lineage commitment in human BM.

Article Snippet: Next, BMMNC were either cryopreserved in FBS with 10% of DMSO or previously enriched for CD34 + (CD34 MicroBead Kit, Miltenyi Biotec) before cryopreservation.

Techniques: Quantitative Proteomics, Expressing, Construct, Activity Assay, Gene Expression

Journal: Cell Reports Methods

Article Title: Integrating transcription-factor abundance with chromatin accessibility in human erythroid lineage commitment

doi: 10.1016/j.crmeth.2022.100188

Figure Lengend Snippet:

Article Snippet: Next, BMMNC were either cryopreserved in FBS with 10% of DMSO or previously enriched for CD34 + (CD34 MicroBead Kit, Miltenyi Biotec) before cryopreservation.

Techniques: Mass Cytometry, Software

Journal: Journal for Immunotherapy of Cancer

Article Title: ESPEC-SUIT: a versatile and robust platform to identify and track antigen-specific T cell receptors in patients with cancer

doi: 10.1136/jitc-2025-012216

Figure Lengend Snippet: Epitope-specific expansion cultures (ESPEC) efficiently and specifically expand desired major histocompatibility complex (MHC) I-restricted and MHC II-restricted T cell responses. ( A ) Overview of workflow for ESPEC; ( B ) overview of analyzed patients and antigens used for ESPEC-SUIT; ( C ) frequency of antigen-specific T cell responses as determined by interferon (IFN)γ Enzyme-Linked ImmunoSpot (ELISpot) in peripheral blood mononuclear cell (PBMC) ( ex vivo , gray, 300 000 cells/well) or post-ESPEC samples (colored, 25 000–50 000 cells/well) for patients with glioma undergoing vaccination with shared (neo-1, n=22; neo-2, n=5) or individual MHC II-restricted neoepitopes (i1-3, n=2 patients, n=3 antigens); open symbols (gray/colored) indicate lack of antigen-specific T cell response ( ex vivo or post-ESPEC); raw data inlays show well images of a representative neo-1 vaccinated patient post-ESPEC for no peptide (‘no pep’) and neo-1-stimulated cultures (top) versus ex vivo (bottom), re-stimulated without antigen (ns) or with neo-1 peptide; ( D ) frequency of antigen-specific T cell responses determined by IFNγ ELISpot in post-ESPEC samples (100 000–250 000 cells/well) for patients with liposarcoma ( i4 ), melanoma ( i5 ) or colorectal cancer ( i6 ) not undergoing vaccination at time of blood sampling. i4 and i6 were in the past vaccinated with long peptides encompassing mutations also contained in short (9–11-mers, MHC I-presented) or long (>20 mer, MHC II-presented) peptides used in ESPEC (‘targeted’, dark gray). Long peptides used for stimulation contained amino acid sequences spanning point mutations or resulting from fusions/missense mutations; short peptides were mutation-derived epitopes predicted to bind the patient’s MHC I molecules; ESPEC also included antigens not targeted by vaccination (‘non-targeted’, light gray); numbers above datasets indicate no. of antigens investigated; heatmap of IFNγ ELISpot spot-forming units (SFU) in ( E ) cultures stimulated with patient-individual ( i6 ) 20-mer peptides (x-axis) after re-stimulation (y-axis) without antigen (‘no peptide’), with the antigen-of-interest (diagonal) or control antigen (MOG), or ( F ) healthy donor (HD1)-derived cultures (x-axis) after ESPEC with four different peptide pools (pp1–pp4) of short (MHC I-restricted, ‘( I )’) or long (MHC II-restricted, ‘(II)’) recall antigens. Post-ESPEC, cells were re-stimulated (y-axis) with single antigens present (circles in heatmap) or absent from the pool; schematic graph to left of heatmap shows strength of the response to the antigens when used as single peptides in ESPEC; intracellular cytokine staining of tumor necrosis factor (TNF)α production in living/CD3 + /CD4 + T cells after re-stimulation of 1×10 6 ( ex vivo ) or 0.4×10 6 (post-ESPEC) cells/condition in representative patients undergoing vaccination with ( G ) neo-1 or ( H ) two patient-individual neoepitopes (neo-X, neo-Y). Panels show the response pre-ESPEC (‘ ex vivo’ ), and post-ESPEC in control stimulated cultures (‘no peptide’) or cultures stimulated with antigen-of-interest (‘neo-1/X/Y’, as indicated above the panel), gated on CD4 + T cells (ID15 and i1-2 top panel) vs CD8 + T cells (i1-2, bottom panel). Histograms show responses to re-stimulation with various peptides: CMV (cytomegalovirus) peptide is included as a control for the unspecific expansion of endogenous T cell responses, wt-1 is included to assess cross-reactivity of neo-1-stimulated cells and neo-X/Y are specificity controls for recently stimulated T cells. APC, antigen-presenting cell; TCR, T cell receptor; neo, neoepitope.

Article Snippet: On days 13–15, cells were harvested, counted and used for (i) TCR repertoire analysis (1×10 6 cells per culture pelleted and cryopreserved after optional enrichment of CD4 + or CD8 + T cells using magnetic bead-based positive selection (human CD4/CD8 microbeads, 130-45-101/201, Miltenyi)), (ii) scV(D)J sequencing (up to 1×10 6 cells cryopreserved in DMSO-containing freezing medium) or (iii) functional assessment of epitope-based expansion of relevant T cell populations after overnight resting in cytokine-free media.

Techniques: Immunopeptidomics, Enzyme-linked Immunospot, Ex Vivo, Sampling, Mutagenesis, Derivative Assay, Control, Staining

Journal: Journal for Immunotherapy of Cancer

Article Title: ESPEC-SUIT: a versatile and robust platform to identify and track antigen-specific T cell receptors in patients with cancer

doi: 10.1136/jitc-2025-012216

Figure Lengend Snippet: Epitope-specific expansion cultures (ESPEC)-induced changes in T cell receptor (TCR) repertoire composition. TCR repertoire ( A ) diversity (mean±SD) and ( B ) clonality (mean±SD) of post-ESPEC samples (n=80 from 32 individuals) expanded without peptide (‘ctrl’) or under antigen-of-interest (‘stim’); note that some donors were stimulated with multiple different peptides or peptide pools in separate cultures, thus contributing multiple post-ESPEC samples and a single ‘ctrl’. # **p≤0.01, Mann-Whitney U test; two-tailed; ( C ) mean percentage of repertoire occupied by clones with rank 1–100, 101–1000 or 1001–10 000 in post-ESPEC samples expanded without peptide (‘ctrl’, n=32) or with antigen (‘stim’, n=80, as some donors were stimulated with multiple antigens) # ; ( D ) clonality in antigen-of-interest (‘stim’) or control stimulated cultures (‘ctrl’), where ESPEC induced an increase in the ELISpot response to neo-1 stimulation of <10-fold (n=7) or >10-fold (n=11) # ; ( E ) fold expansion of the 10 largest clonotypes post-ESPEC compared with their pre-ESPEC frequency in two healthy donors (HD2, HD3) and patient i4 stimulated with MHC I-restricted (light gray) or MHC II-restricted (dark gray) recall antigens (top panel), and three patients ( i3–i4, i6 ) stimulated with short (light gray) or long (dark gray) peptides encoding patient-individual neoepitopes, including a patient-individual gene fusion targeted by peptide vaccination ( i3 ). Full repertoire data were evaluated to avoid loss of small clones by down-sampling. Numbers next to the y-axis indicate number of top 10 post-ESPEC clonotypes that were below the limit of detection pre-ESPEC; ( F ) fold expansion of the 10 largest clonotypes post-ESPEC compared with their pre-ESPEC frequency in all neoepitope ESPEC cultures (n=63), sorted by antigen type and magnitude of the post-ESPEC ELISpot response # . Symbol hues indicate cultures enriched for CD4 + (teal, mint, light green) or CD8 + (dark green) T cells prior to repertoire sequencing. Numbers above the x-axis indicate the number of top 10 post-ESPEC clonotypes that were below the limit of detection pre-ESPEC; frequency of the 10 largest clonotypes post-ESPEC compared with their pre-ESPEC (baseline (‘BL’)) frequency for representative cultures exposed to ( G ) short and ( H ) long peptides and enriched for the relevant T cell subset # . **P≤0.01, Wilcoxon signed-rank test, two-tailed). # TCRβ deep-sequencing data were down-sampled to 10 000 TCR counts (panel A–D, F–H ).

Article Snippet: On days 13–15, cells were harvested, counted and used for (i) TCR repertoire analysis (1×10 6 cells per culture pelleted and cryopreserved after optional enrichment of CD4 + or CD8 + T cells using magnetic bead-based positive selection (human CD4/CD8 microbeads, 130-45-101/201, Miltenyi)), (ii) scV(D)J sequencing (up to 1×10 6 cells cryopreserved in DMSO-containing freezing medium) or (iii) functional assessment of epitope-based expansion of relevant T cell populations after overnight resting in cytokine-free media.

Techniques: MANN-WHITNEY, Two Tailed Test, Clone Assay, Control, Enzyme-linked Immunospot, Sampling, Sequencing

Journal: Journal for Immunotherapy of Cancer

Article Title: ESPEC-SUIT: a versatile and robust platform to identify and track antigen-specific T cell receptors in patients with cancer

doi: 10.1136/jitc-2025-012216

Figure Lengend Snippet: Tracing and characterization of antigen-reactive clonotypes in peripheral blood and tissue. ( A–B ) Representative data of the cumulative frequency of epitope-specific expansion cultures (ESPEC)-derived candidate T cell receptors (TCRs) (bars, left y-axis) and the ex vivo interferon (IFN)γ Enzyme-Linked ImmunoSpot (ELISpot) responses against neo-1 peptide (background subtracted, dotted line, right y-axis) at the relevant time points in peripheral blood of two neo-1 vaccinated patients. In ( B ), clonotypes with in vitro validated neo-1 reactivity are highlighted in teal, untested or unreactive candidates are shown in gray; for comparison between peripheral blood mononuclear cell (PBMC) samples, TCRβ deep-sequencing data was down-sampled to 50 000 counts; blood was collected before vaccination and after 1–7 administrations of long neo-1 encoding peptide (A: vaccination on week ( W ) 0, 2, 4, 10; B: vaccination on W0, 2, 4, 9, 17); number ( C ) and cumulative frequency ( D ) of ESPEC-derived candidate clonotypes (teal) versus all other clonotypes in tumors of neo-1 immunized patients (n=4, 5 tumors), and patient i3 (two tumors), who received a personalized, fusion-spanning peptide vaccine; tumor tissue was collected after eight vaccinations; raw ( E ) and quantified (mean±SD) ( F ) neo-1-specific IFNγ ELISpot response in minimally cultured tumor-infiltrating lymphocytes from two patients with glioma treated with neo-1 long peptide vaccine, after stimulation of 5000 cells/well with no peptide (ctrl) or neo-1 20-mer, and ( G ) cumulative frequency of neo-1 ESPEC-derived candidate clonotypes detected in the tumor-infiltrating lymphocyte (TIL) TCR repertoire of these patients as assessed by bulk (ID7) or single-cell (ID8) TCR sequencing; uniform manifold approximation and projection highlighting gene expression profiles of tumor-infiltrating lymphocytes in n=3 neo-1 vaccinated patients with TCRβ CDR3 sequences ( H ) derived from in vitro validated neo-1-reactive clonotypes (n=70 in n=827 cells) or ( I ) shared (‘candidates’, n=246 clonotypes in n=1270 cells) and not shared (‘other’, n=6519 clonotypes in n=11 779 cells) with ESPEC candidates selected from neo-1-stimulated cultures of the respective patient.

Article Snippet: On days 13–15, cells were harvested, counted and used for (i) TCR repertoire analysis (1×10 6 cells per culture pelleted and cryopreserved after optional enrichment of CD4 + or CD8 + T cells using magnetic bead-based positive selection (human CD4/CD8 microbeads, 130-45-101/201, Miltenyi)), (ii) scV(D)J sequencing (up to 1×10 6 cells cryopreserved in DMSO-containing freezing medium) or (iii) functional assessment of epitope-based expansion of relevant T cell populations after overnight resting in cytokine-free media.

Techniques: Derivative Assay, Ex Vivo, Enzyme-linked Immunospot, In Vitro, Comparison, Sequencing, Cell Culture, Gene Expression