Journal: Journal of Translational Medicine

Article Title: Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality

doi: 10.1186/s12967-026-07700-6

Figure Lengend Snippet: Ex vivo expansion of HSPCs in A83-01, pomalidomide, and UM171 (APU). ( A ) Experimental scheme of the 7-day ex vivo expansion protocol. CD34 + CD38 − CD45RA − cells from umbilical cord blood (UCB) were seeded in low cell density for HSPC expansion and analyzed via flow cytometry for their HSC immune phenotype. ( B ) Representative plots for the gating strategy of CD34 + CD38 − CD45RA − CD90 + EPCR + cells in SFT3 (basal medium), UM171, and APU. ( C ) Surface marker expression of CD34, CD38, CD45RA, CD90, and EPCR in N = 3 UCB donors after 7-day expansion of CD34 + CD38 − CD45RA − cells in SFT3, UM171, and APU with 2 µM or 0.2 µM pomalidomide (Poma). ( D ) Expansion of the total cell number in one 96-well counted with Casy cell counter. ( E ) Fold change of expanded ST-HSCs after 7-days. The number of expanded CD34 + CD38 − CD45RA − CD90 + cells was divided by 1,000 (seeded cell number) and then normalized to SFT3. ( F ) Percentage of CD34 + CXCR4 + HSPCs after expansion. The mean is displayed with the SD by the error bars. Statistical analysis in D-F was performed with one-way ANOVA and Tukey’s test for multiple comparisons in N = 4 UCB donors (* = p < 0.05, ** = p < 0.01)

Article Snippet: Thus, CD34 + -enriched HSPCs were thawed and blocked with Fc receptor blocking reagent (Miltenyi Biotec) before staining with antibodies against CD34 (APC, clone HG12, catalog #345804, BD Biosciences, Franklin Lakes, NJ, Unites States), CD38 (FITC, clone HB7, catalog #11-0388-42, Thermo Fisher Scientific, Waltham, MA, USA), and CD45RA (BV605, clone HI100, catalog #304134, BioLegend, San Diego, CA, USA).

Techniques: Ex Vivo, Flow Cytometry, Marker, Expressing

Journal: Journal of Translational Medicine

Article Title: Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality

doi: 10.1186/s12967-026-07700-6

Figure Lengend Snippet: Single-cell RNA sequencing (scRNA seq) analysis reveals expression of HSC signature genes in APU-exp HSPCs. ( A ) Surface marker expression of CD34, CD38, CD45RA, CD90, and EPCR after 7-day expansion in SFT3 or SFT3 supplemented with single (A, P, U), minus-one (AP, AU, PU), and complete APU (APU with 2 µM or 0.2 µM Poma) compound conditions. ( B ) Experimental scheme for the scRNA seq experiment using the 10X Genomics Chromium technology and hashtag oligo (HTO)-labeled antibodies for sample multiplexing. UCB-derived CD34 + CD38 − CD45RA − HSPCs were analyzed uncultivated, 24 h cultivated in SFT, SFT + APU, or SFT + APU_0.2, and 7-day expanded in the conditions described in ( A ). ( C ) Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) displaying the integrated data of all samples with cluster annotation: Hematopoietic stem cells/multipotent progenitors (HSCs/MPPs), early and late cycling megakaryocytic-erythroid-mast cell progenitors (cMEMPs), granulocytes (neutrophils, eosinophils, basophils/mast cells), mast cells, monocytes and macrophages (Mono’s/Neutro’s), cycling MPPs (cMPPs), and cycling dendritic cells (cDCs). ( D ) Dot plot showing the average expression of the respective marker genes used in cluster annotation

Article Snippet: Thus, CD34 + -enriched HSPCs were thawed and blocked with Fc receptor blocking reagent (Miltenyi Biotec) before staining with antibodies against CD34 (APC, clone HG12, catalog #345804, BD Biosciences, Franklin Lakes, NJ, Unites States), CD38 (FITC, clone HB7, catalog #11-0388-42, Thermo Fisher Scientific, Waltham, MA, USA), and CD45RA (BV605, clone HI100, catalog #304134, BioLegend, San Diego, CA, USA).

Techniques: Single Cell, RNA Sequencing, Expressing, Marker, Labeling, Multiplexing, Derivative Assay

Journal: Journal of Translational Medicine

Article Title: Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality

doi: 10.1186/s12967-026-07700-6

Figure Lengend Snippet: Effect of APU on HSPCs after 24 h cultivation on the single-cell level. ( A ) UMAP of uncultivated and 24 h-cultivated HSPCs and their individual cluster abundances in the bar plot below. ( B ) Feature plots of the HSC signature genes HLF , MECOM , NKAIN1 , PROM1 , AVP , and CD34 . ( C) Dotplot comparing the average expression of HSC signature genes among the 24 h-cultivated HSPCs. ( D ) Volcano plot displaying the log2 fold changes (FC) and the -log10 p values of DEGs between HSPCs cultivated for 24 h in SFT + APU (APU_24h) compared to SFT (SFT_24h). Significant ( p > 0.05, Wilcoxon Rank Sum test) genes with an FC > 1 are colored in green (up in APU_24h) or yellow (up in SFT_24h). ( E ) Gene set enrichment analysis (GSEA) on the DEGs of APU_24h vs. SFT_24h using HSC-related gene sets from the Molecular Signatures Database (MSigDB), showing the normalized enrichment score (NES). ( F ) GSEA on the DEGs of APU_24h vs. SFT_24h and of SFT3 + APU_7d vs. SFT3_7d (HSC/MPP cluster) using TGFβ-related gene sets from the MSigDB

Article Snippet: Thus, CD34 + -enriched HSPCs were thawed and blocked with Fc receptor blocking reagent (Miltenyi Biotec) before staining with antibodies against CD34 (APC, clone HG12, catalog #345804, BD Biosciences, Franklin Lakes, NJ, Unites States), CD38 (FITC, clone HB7, catalog #11-0388-42, Thermo Fisher Scientific, Waltham, MA, USA), and CD45RA (BV605, clone HI100, catalog #304134, BioLegend, San Diego, CA, USA).

Techniques: Single Cell, Expressing

Journal: Journal of Translational Medicine

Article Title: Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality

doi: 10.1186/s12967-026-07700-6

Figure Lengend Snippet: Increased lentiviral transduction efficiency in APU-exp cells. ( A ) Graphical scheme of the combined transduction (td) and expansion protocol for UCB-derived CD34 + cells. Td was performed with 1 × 10 5 CD34 + 24 h post-thawing with a self-inactivating lentiviral vector encoding for mCherry driven by the CBX3.EFS promoter (SIN-LV.CBX3.EFS.mCherry) in APU/SFT3 medium supplemented with protamine sulfate and Synperonic ® F-108 in a U-bottom 96-well plate. On day 1 post-td, HSPCs were seeded for expansion. Td efficiency and stem cell phenotype were determined on day 6 post-td, and vector copy number (VCN) was determined on day 8 post-td. Each donor replicate of expanded cells was split for transplantation into three mice. ( B ) Number of cells after thawing CD34 + cells from N = 3 UCB donors (day − 1), the next day (day 0), and after expansion (day 6 post-td) counted with Casy. ( C ) HSPC immune phenotype of expanded cells 6 days post-td. Expression of CD34 + , CD34 + CD38 − CD45RA − CD90 + (labeled CD90 + ), and CD34 + EPCR + in expanded HSPCs. ( D ) Td efficiency in the bulk cell population, in CD34 + HSPCs, and CD34 + CD38 − CD45RA − CD90 + EPCR + (labeled as LT-HSC). ( E ) VCN per diploid cell determined by qPCR. Bars indicate the mean of n = 3 UCB donors with the SD displayed by the error bars; Statistical significances were determined by paired t-test; p < 0.05= *, p < 0.0001= ****

Article Snippet: Thus, CD34 + -enriched HSPCs were thawed and blocked with Fc receptor blocking reagent (Miltenyi Biotec) before staining with antibodies against CD34 (APC, clone HG12, catalog #345804, BD Biosciences, Franklin Lakes, NJ, Unites States), CD38 (FITC, clone HB7, catalog #11-0388-42, Thermo Fisher Scientific, Waltham, MA, USA), and CD45RA (BV605, clone HI100, catalog #304134, BioLegend, San Diego, CA, USA).

Techniques: Transduction, Derivative Assay, Plasmid Preparation, Transplantation Assay, Expressing, Labeling

Journal: Journal of Translational Medicine

Article Title: Ex vivo long-term expansion of human hematopoietic stem and progenitor cells as a tool for modeling vector integration sites and clonality

doi: 10.1186/s12967-026-07700-6

Figure Lengend Snippet: APU_0.2 allows long-term cultivation of CD34 + cells up to 5 weeks after lentiviral transduction. ( A ) Experimental setup of transducing expanded HSPCs. Purified CD34 + CD38 − CD45RA − cells were expanded for 7 days either in SFT3 or APU_0.2 medium before transduction (td) with the SIN-LV.SF.eGFP (SF) or the SIN-LV.EFS.eGFP (EFS) vector. 24 h post-td, HSPCs were either transplanted into immunocompromised mice or further cultivated. Long-term culture allowed downstream analyses. ( B ) Expansion factor after 7-day culture in SFT3 or APU_0.2 medium determined by Casy. Paired t-test revealed non-significant (ns = p > 0.05) differences. ( C ) Flow cytometric analysis of eGFP expression on day 7 post-td to determine the td efficiency. ( D ) Vector copy number (VCN) per diploid genome was determined via ddPCR. ( E ) Expansion factor over 5 weeks post-td either in SFT3 or APU_0.2 medium. Cell numbers were determined by Casy and divided by the seeded cell number from the week before. Paired t-test over the whole period of time between APU_0.2 and SFT3 to assess the expected mean difference (diff.) between the groups, the 95% confidence interval (CI), and the p value. ( F ) Microscopic images 3 weeks post-td. Imaging was performed with CellCyteX using 10x magnification. Scale bar length indicates 100 μm. ( G-H ) Monocytic markers (CD11b, CD14, CD33) were determined via flow cytometry 3, 4, and 6 weeks post-td of HSPCs cultivated in SFT3 ( G ) or APU_0.2 ( H ). ( I ) Dim and bright CD11b expression in HSPCs cultivated for 3 weeks post-td. Exemplary gating strategy of the CD11b + bright CD14 + population for SFT3 cultivated cells. ( J ) Percentage of CD34 + cells during the long-term culture in SFT3 or APU_0.2 up to 5 weeks post-td. Two-way ANOVA with Tukey’s multiple comparisons test. Individual values are biological replicates with N = 3 UCB donors. Error bars indicate the mean ± SD

Article Snippet: Thus, CD34 + -enriched HSPCs were thawed and blocked with Fc receptor blocking reagent (Miltenyi Biotec) before staining with antibodies against CD34 (APC, clone HG12, catalog #345804, BD Biosciences, Franklin Lakes, NJ, Unites States), CD38 (FITC, clone HB7, catalog #11-0388-42, Thermo Fisher Scientific, Waltham, MA, USA), and CD45RA (BV605, clone HI100, catalog #304134, BioLegend, San Diego, CA, USA).

Techniques: Transduction, Purification, Plasmid Preparation, Expressing, Imaging, Flow Cytometry

Journal: bioRxiv

Article Title: Interleukin-6 restricts pre-thymic T cell lineage commitment of progenitors driving loss of SIV control

doi: 10.64898/2026.01.29.702123

Figure Lengend Snippet: a, Schematic illustrating thymus-seeding progenitors (TSPs) arising from double-negative (DN; CD4⁻CD8⁻) CD34⁺CD7⁺ hematopoietic stem and progenitor cells (HSPCs) in the bone marrow and their migration to the thymus, where they undergo stepwise differentiation through CD4⁺CD3 − immature single-positive (ISP), CD4⁺CD8⁺ double-positive (DP), and single-positive (SP) CD4⁺ or CD8⁺ T-cell stages. b-c, Representative flow-cytometry plots showing (DN)CD34⁺CD7⁺ HSPCs in bone marrow (b) and thymus (c), with corresponding fluorescence-minus-one (FMO) controls. d, Frequency of (DN)CD34⁺CD7⁺ TSPs among bone-marrow and thymic HSPCs. e, Absolute numbers of (DN)CD34⁺CD7⁺ HSPCs per 10 6 total bone marrow mononuclear cells and thymocytes analyzed. f–i, Pearson correlation analyses between matched (N=8) bone-marrow and thymus derived CD4 − CD8 − double negative CD34 + progenitor populations, comparing frequencies of CD7 expressing (f), and non-expressing (g) DN CD34⁺ HSPCs. h, Pearson correlation between bone marrow derived CD34⁺CD38 − CD7⁺ (DN1) TSPs and thymus derived (DN)CD34+ (h), and (DN)CD34+CD7+TCF1+ (i) progenitors. Pearson correlation coefficients (r) and P values are shown. j, Schematic of the previously described Rhesus-specific nonanimal model of thymopoiesis known as Rhesus-artificial thymic organoid (RhATO) system , seeded with CD3 − (DN)CD34⁺CD7⁺ TSPs. k, Representative flow-cytometry plots illustrating T cell lineage committed differentiation intermediates representing frequencies of ISPs, and DPs early thymocyte intermediates, mature CD3⁺ T cells, as well as CD4⁺ and CD8⁺ single positive T cells, analyzed at week 4 of organoid establishment as previously described (k). l, Bar graph showing quantification of the frequencies of indicated thymocyte subsets generated in the RhATO-system (N=6 RhATOs). m, Schematic depicting uninfected (SIV⁻; N=15) and SIV-infected (SIV⁺; N=12) rhesus macaques (RMs) used to analyze bone marrow derived progenitors. n, Representative flow-cytometry plots showing (DN)CD34⁺CD7⁺ T cell committed progenitors in bone marrow of SIV⁻ and SIV⁺ RMs. o, Bar graph showing quantitation of the frequency of (DN)CD34⁺CD7⁺ TSPs among bone-marrow HSPCs in SIV⁻ and SIV⁺ RMs. p, Absolute numbers of (DN)CD34⁺CD7⁺ T-cell committed progenitors per 10⁶ bone-marrow mononuclear cells (BMMCs) analyzed. q, Absolute numbers of (DN)CD34⁺ HSPCs per 10⁶ BMMCs analyzed. r, Frequency of CD34⁺CD38 − CD7⁺ (DN1) TSPs among bone-marrow (DN)CD34+CD7+ progenitors in SIV⁻ and SIV⁺ animals. s, Experimental design showing RMs (N=6) infected with 300 TCID 50 SIVmac239M via intravenous route. Bone marrow aspirates (red dots) and blood (black dots) collected at the indicated timepoints starting with week 0 of infection. t, Line graph showing longitudinal plasma viral load (PVL) at the indicated timepoints. Geomean PVL indicated in black and individual data indicated in grey. u-v, Line graphs showing longitudinal frequencies of (DN)CD34⁺CD7⁺ (u) and CD34⁺CD38 − CD7⁺ (DN1) TSPs (v) TSPs at baseline (week 0) and at week 2, 4, 12, and 16 following SIV infection. w, Representative flow-cytometry plots and bar graph showing frequencies of T cells generated in RhATO cultures using 5000 CD3 − CD34 + HSPCs per RhATO isolated from bone marrow of RMs at week 16 of infection. x, Pearson correlation analysis between fold decrease of CD34 + CD38 − CD7 + (DN1) TSP population at week 2 of infection and frequency of T cells generated by CD3 − CD34 + HSPCs in RhATO culture. y–z, Pearson correlation analyses between total plasma viral burden until week 16 post infection represented as area under curve and fold decrease of CD34 + CD38 − CD7 + (DN1) TSP (y), and frequency of T cells generated in RhATO (z). Each symbol represents one animal. Statistical significance was determined using Mann Whitney unpaired two-tailed t-tests for unrelated data sets, Pearson correlation, or paired Wilcoxon test for related data sets; (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).

Article Snippet: Isolation of human CD3-CD34+ HSPC: Total CD34+ HSPCs (Cat#BM34C-5) purchased from Charles River Laboratory Inc, Wilmington, MA, USA, were thawed and directly stained with live/dead stain, anti-CD3, and anti-CD34 antibodies.

Techniques: Migration, Flow Cytometry, Fluorescence, Derivative Assay, Expressing, Generated, Infection, Quantitation Assay, Clinical Proteomics, Isolation, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: Interleukin-6 restricts pre-thymic T cell lineage commitment of progenitors driving loss of SIV control

doi: 10.64898/2026.01.29.702123

Figure Lengend Snippet: a , Longitudinal plasma viral load (PVL) in SIV-infected rhesus macaques (RMs) measured before infection (D0) and at week (Wk) 2, 4 and 16 post SIV infection (N=6). b–f , Line graphs and corresponding heatmaps representing longitudinal plasma concentrations of IL-6 ( b ), IL-15 ( c ), TNF-β ( d ) IL-1β ( e ) and IP-10 (CXCL10) ( f ) across the same time points. ( g) , Pearson correlation matrices summarizing associations between CD34⁺CD38 − CD7⁺ (DN1) thymic seeding progenitors (TSPs) attrition, plasma viral load and circulating cytokines and chemokines during peak viremia (week 2 post SIV). Pearson correlation coefficients (r) is shown. ( h–k) , Pearson correlation between peak PVL and corresponding plasma concentrations of IL-6 ( h ), IL-15 ( i ), TNF-β ( j ) IL-1β ( k ) at week 2 post infection. l, Pearson correlation between (DN1) TSP attrition and plasma IL-6 concentrations at peak viremia (week 2) post infection. (m-n), Representative flow-cytometry plots illustrating RhATO-derived T cell developmental ontogeny from bone marrow CD3 − CD34 + HSPCs of RMs isolated at week 16 of infection, culture analyzed at week 4 of establishment. Flow plots show differentiation stages of thymocytes including CD4 + CD3 − immature single positive (ISP) early thymocyte, CD4 + CD8 + double positive (DP) intermediate thymocyte, (m) and mature CD3⁺ T cells (n). o-q, Pearson correlation analyses between plasma IL-6 at week 16 of infection and RhATO derived frequencies of ISPs (o), DPs (p), and CD3+ T cells (q) stages. r, Pearson correlation analyses between plasma IL-6 at week 16 of infection and frequency of CD3+ T cells in blood of RMs at week 16 post infection. s , Pearson correlation between fold decrease in frequency of bone marrow (DN1) TSPs and plasma IP-10 concentrations. Each symbol represents one animal; lines indicate longitudinal measurements from the same animal. Heat-map color scales indicate relative concentrations or viral load. Linear regression lines, Pearson correlation coefficients (r) and P value calculated by paired Wilcoxon test are shown where appropriate; (*P < 0.05; **P < 0.01).

Article Snippet: Isolation of human CD3-CD34+ HSPC: Total CD34+ HSPCs (Cat#BM34C-5) purchased from Charles River Laboratory Inc, Wilmington, MA, USA, were thawed and directly stained with live/dead stain, anti-CD3, and anti-CD34 antibodies.

Techniques: Clinical Proteomics, Infection, Flow Cytometry, Derivative Assay, Isolation

Journal: bioRxiv

Article Title: Interleukin-6 restricts pre-thymic T cell lineage commitment of progenitors driving loss of SIV control

doi: 10.64898/2026.01.29.702123

Figure Lengend Snippet: a, Cartoon illustrating early T cell lineage committed differentiation steps from CD4⁻CD8⁻CD34⁺CD38⁻CD7⁺ (DN1) TSPs to CD4 + CD3 − immature single-positive (ISPs) thymocyte stage, highlighting early phenotypic changes during T cell committed differentiation process. b, Experimental schematic illustrating isolation of CD3 − CD34 + hematopoietic stem and progenitor cells (HSPCs) from bone-marrow of SIV-uninfected (SIV⁻; N = 15) and SIV-infected (SIV⁺; N = 12) rhesus macaques (RMs) and their differentiation to ISP thymocyte in the rhesus-artificial thymic organoid (RhATO) system. c, Representative flow-cytometric plots showing frequencies of ISPs and corresponding CD45⁺ lymphocytes generated in RhATO culture at week 1 of culture as previously described. d, Bar graph showing quantitation of the frequency of ISP thymocytes for SIV⁻ (N=31 RhATOs) and SIV⁺ (N=34 RhATOs) conditions. e, Bar graph showing corresponding frequency of CD45⁺ lymphocytes. f, Bar graph showing ratio of the frequencies of ISP thymocytes to CD45⁺ lymphocytes generated in RhATO system. g, Schematic showing longitudinal ex-vivo tracking of the T cell lineage committed differentiation potential of the CD3 − CD34 + HSPCs isolated from bone marrow of six RMs at the indicated timepoints (red dots) post SIV infection. h, Representative flow-cytometric plot showing frequencies of the ISP thymocytes generated in RhATOs. Each plot represents each timepoint post infection T cell lineage committed potential of HSPCs were evaluated. i, Line graph showing geomean (dark green line) and individual data points (lighter green lines) of the frequencies of CD4⁺CD3⁻ ISP thymocytes generated in RhATOs from bone-marrow HSPCs collected at the indicated time points following SIV infection. j, Pearson correlation between drop in T cell lineage committed differentiation potential of bone marrow derived HSPCs (as estimated by frequency of ex-vivo generated ISPs in RhATO cultures at week 2 compared with day 0 post infection) and plasma IL-6 concentrations at week 2 post infection. k, Representative flow-cytometric plots identifying Lin⁻CD34⁺CD38⁻CD90⁻CD45RA⁺ lymphoid-primed multipotent progenitors (LMPPs) in bone marrow at baseline (SIV D0), week 2 and week 16 post-infection. l, Line graph showing geomean (dark blue) and individual values (lighter blue lines) frequencies of LMPPs in bone marrow at day 0, week 2 and 16 post infection. Each symbol represents an individual animal. Data are presented as geomean ± geomean standard deviation. Statistical significance was determined using Mann Whitney unpaired two-tailed t-tests for unrelated data sets, Pearson correlation, or paired Wilcoxon test for related data sets; (*P < 0.05; ****P < 0.0001; ns, not significant).

Article Snippet: Isolation of human CD3-CD34+ HSPC: Total CD34+ HSPCs (Cat#BM34C-5) purchased from Charles River Laboratory Inc, Wilmington, MA, USA, were thawed and directly stained with live/dead stain, anti-CD3, and anti-CD34 antibodies.

Techniques: Isolation, Infection, Generated, Quantitation Assay, Ex Vivo, Derivative Assay, Clinical Proteomics, Standard Deviation, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: Interleukin-6 restricts pre-thymic T cell lineage commitment of progenitors driving loss of SIV control

doi: 10.64898/2026.01.29.702123

Figure Lengend Snippet: a , Representative flow-cytometry plots showing frequencies of DN CD34⁺CD7 + T cell committed progenitors in RhATO cultures supplemented without (positive control) or with increasing concentrations of IL-6 (0.5, 5 or 50 ng ml⁻¹), analyzed at week 1 of culture establishment with bone marrow derived CD3 − CD34 + HSPCs from SIV naïve rhesus macaques (RMs) as previously described. DN progenitors were identified based on CD4 − CD8 − double negative (DN), CD34 and CD7 expression; numbers indicate frequencies within the gated populations. b , Bar graph showing quantification of the frequency of (DN)CD34⁺CD7⁺ TSPs across IL-6 concentrations, demonstrating a dose-dependent reduction in T cell progenitors. c, Schematic of the IL-6 receptor blockade experimental design. Bone marrow derived CD3 − CD34⁺ HSPCs from SIV naive RMs were FACS sorted and cultured RhATO system in the absence of IL-6 (grey), presence of IL-6 (green), and in the presence of IL-6 receptor binding antibody + IL-6 (blue), as indicated. d , Representative flow-cytometry plots showing (DN)CD34⁺CD7 + T cell progenitors in RhATO cultures at week 1 of culture establishment with bone marrow derived CD3 − CD34⁺ HSPCs from SIV naive RMs for above-described treatment conditions. e , Bar graph showing quantitation of the frequency of (DN)CD34⁺CD7⁺ progenitors across treatment conditions (N=6 RhATOs). f , Representative flow-cytometry plots showing T cell lineage committed differentiation of CD3 − CD34⁺ HSPCs marked by increase in the frequency of CD4 + CD3 − immature single positive thymocytes (ISPs) in RhATO. ISPs (early thymocyte intermediate) were identified based on CD45 + CD8 − CD3 − and CD4 + expression; numbers indicate frequencies within the gated populations. g , Bar graph showing quantification of the frequency of CD4 + CD3 − ISPs across treatment conditions. h , Schematic illustrating key early intermediates along the T cell committed differentiation (CD4 − CD8 − double negative, DN1 (CD38 − ), DN2/3(CD38 + ), immature single-positive thymocyte; ISP) and associated marker expression (CD34, CD7, CD38 and CD4). Arrows indicate relative changes in marker expression under IL-6⁻ and IL-6⁺ treatment conditions. i , Representative flow-cytometry plots showing frequencies of CD34 + CD38 − and CD34 + CD38 + fractions within CD4 − CD8 − (DN) HSPCs (SIV naïve) cultured in RhATO-system in the absence or presence of IL-6. J-k , Bar graphs showing quantitation of the frequencies of (DN)CD34⁺CD38⁻ (j) and (DN)CD34⁺CD38⁺(k) progenitors under IL-6⁻ and IL-6⁺ treatment conditions (N = 9 RhATOs). l , Representative flow-cytometry plots showing early T cell lineage committed differentiation to CD34⁺CD38 + CD7 + (DN2) TSPs in RhATO cultures under IL-6⁻ and IL-6⁺ treatment conditions (N = 9 RhATOs). m , Bar graph showing quantitation of the frequency of CD34⁺CD38 + CD7+ (DN2) TSPs under IL-6⁻ and IL-6⁺ treatment conditions. n-r , Analysis of the impact of IL-6 exposure on expansion of CD34+CD38+CD7+ (DN2) TSP intermediate in a human-specific nonanimal model of thymopoiesis. n, Bar graph showing quantitation of (DN)CD34⁺CD7⁺ T cell progenitors under IL-6⁻ (N = 6 hATOs) and IL-6⁺ (N = 18 hATOs) conditions. o, Representative flow-cytometry plots showing CD38 expression within CD34⁺CD7⁺ DN progenitors in hATO culture under IL-6⁻ and IL-6⁺ conditions. p-q , Bar graph showing quantitation of the frequencies of CD34⁺CD38⁻ (p) and CD34⁺CD38⁺ (q) progenitors within CD4 − CD8 − (DN) CD34 + HSPCs (HIV naïve) under IL-6⁻ and IL-6⁺ conditions. r , Bar graph showing quantitation of the frequencies of CD34⁺CD38⁺CD7⁺ (DN2) TSPs under IL-6⁻ and IL-6⁺ conditions. Each point represents an individual organoid. Bar graphs show geomean with geomean standard deviation. Statistical significance was assessed using repeated-measures ANOVA with multiple-comparison correction or Mann Whitney unpaired two-tailed t-tests for unrelated data sets; (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant).

Article Snippet: Isolation of human CD3-CD34+ HSPC: Total CD34+ HSPCs (Cat#BM34C-5) purchased from Charles River Laboratory Inc, Wilmington, MA, USA, were thawed and directly stained with live/dead stain, anti-CD3, and anti-CD34 antibodies.

Techniques: Flow Cytometry, Positive Control, Derivative Assay, Expressing, Cell Culture, Binding Assay, Quantitation Assay, Marker, Standard Deviation, Comparison, MANN-WHITNEY, Two Tailed Test

Journal: bioRxiv

Article Title: Interleukin-6 restricts pre-thymic T cell lineage commitment of progenitors driving loss of SIV control

doi: 10.64898/2026.01.29.702123

Figure Lengend Snippet: a–d, Analysis of early events of T cell lineage committed differentiation of bone marrow CD3 − CD34 + HSPCs in the absence or presence of IL-6 at week 1 of RhATO culture. a, Bar graph showing absolute number of DN CD34⁺CD7⁺ T cell progenitors per 5 × 10³ input HSPCs generated in the absence or presence of IL-6. b, Bar graph showing absolute numbers of DN CD34⁺CD38⁻ progenitors per 5 × 10³ input HSPCs in the absence or presence of IL-6. c, Bar graph showing absolute numbers of DN CD34⁺CD38⁺ progenitors per 5 × 10³ input HSPCs in the absence or presence of IL-6. d, Bar graph showing absolute numbers of CD34⁺CD38⁺CD7⁺ (DN2) TSPs per 5 × 10³ input HSPCs following IL-6 treatment. e–j, Analysis of early events of T cell lineage committed differentiation of human bone marrow derived primary CD3 − CD34 + HSPCs (HIV naïve) in the absence or presence of IL-6 at week 1 of hATO culture. e, Representative flow-cytometry plots showing frequency of (DN)CD34+CD7+ T cell progenitors in the absence or presence of IL-6 at week 1 of hATO culture. g, Bar graph showing absolute number of DN CD34⁺CD7⁺ T cell progenitors per 5 × 10³ input HSPCs generated in the absence or presence of IL-6. h, Bar graph showing absolute numbers of DN CD34⁺CD38⁻ progenitors per 5 × 10³ input HSPCs in the absence or presence of IL-6. i, Bar graph showing absolute number of DN CD34⁺CD38⁺ progenitors per 5 × 10³ input HSPCs in the absence or presence of IL-6. j, Bar graph showing absolute numbers of CD34⁺CD38⁺CD7⁺ (DN2) TSPs generated per 5 × 10³ input HSPCs in the absence or presence of IL-6. Each symbol represents an independent donor or biological replicate. Data are shown as geomean ± geomean s.d. Statistical significance was determined using Mann Whitney unpaired two-tailed t-tests. (**P < 0.01; **P < 0.0001).

Article Snippet: Isolation of human CD3-CD34+ HSPC: Total CD34+ HSPCs (Cat#BM34C-5) purchased from Charles River Laboratory Inc, Wilmington, MA, USA, were thawed and directly stained with live/dead stain, anti-CD3, and anti-CD34 antibodies.

Techniques: Generated, Derivative Assay, Flow Cytometry, MANN-WHITNEY, Two Tailed Test