Journal: bioRxiv

Article Title: Targeting MCL1-driven anti-apoptotic pathways to overcome hypomethylating agent resistance in RAS -mutated chronic myelomonocytic leukemia

doi: 10.1101/2023.04.07.535928

Figure Lengend Snippet: A) UMAP of scDNA and protein-seq data for pooled MNCs isolated from BM samples obtained from a CMML patient with pre-existing KRAS T58K and NRAS G12R mutations at diagnosis (n=1,826) and at BP after HMA therapy failure (n=4,001). BP was not associated with the clonal evolution of these mutations as they both had a VAF of approximately 50% at the onset of the disease. Each dot represents one cell. Cells are clustered based on immunophenotypic markers. Different colors represent cluster identity (left) or origin (right). Mono, monocytes; Ery, erythroblasts; cDC, classical dendritic cells; CD4T, CD4 + T-cells; MyHPC, myeloid hematopoietic progenitor cells; CD8T, CD8 + T-cells; NKC, natural killer cells. (B) Heatmap displaying DNA and protein reads from each sequenced cell type shown in . Colors for protein data correspond to antibody-oligonucleotide intensity signals. High protein expression is depicted in red and low protein expression is depicted in blue. DNA colors correspond to the genotypes for each individual mutation per cell read (wild-type=dark grey, mutant=red, missing=light grey) based on cluster. Percentages correspond to the frequencies of mutant reads within each cluster for a given mutation. C) UMAP of scDNA and protein-seq data for pooled MNCs isolated from BM samples obtained from a CMML patient at diagnosis (n=3,213) and at BP after HMA therapy failure (n=5,342). BP was associated with the clonal evolution of a pre-existing CBL F378Ifs mutation and the acquisition of a previously undetected CBL C384Y mutation. Each dot represents one cell. Cells are clustered based on immunophenotypic markers. Different colors represent cluster identity (left) or origin (right). Mono, monocytes; Ery, erythroblasts; DC, classical dendritic cells; CD4T, CD4 + T-cells; B-cell, B lymphocytes, myeloid hematopoietic progenitor cells; CD8T, CD8 + T-cells; NKC, natural killer cells. (D) Heatmap displaying DNA and protein reads from each sequenced cell type as shown in . Colors for protein data correspond to antibody-oligonucleotide intensity signals. Red indicates high protein expression, and blue indicates low protein expression. Colors for DNA data correspond to the genotype for each individual mutation per cell read (dark grey, wild type; red, mutant; light grey, missing) based on cluster. Percentages correspond to the frequencies of mutant reads within each cluster for a given mutation.

Article Snippet: For cell sorting applications, MNCs were enriched in CD34 + cells using magnetic-activated cell sorting (MACS) with the CD34 Microbead Kit (catalog number #130-046-702, Miltenyi Biotec, Germany) and further purified by fluorescence-activated cell sorting (FACS) as described below.

Techniques: Isolation, Biomarker Discovery, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Targeting MCL1-driven anti-apoptotic pathways to overcome hypomethylating agent resistance in RAS -mutated chronic myelomonocytic leukemia

doi: 10.1101/2023.04.07.535928

Figure Lengend Snippet: (A) UMAP of scRNA-seq data for pooled single Lin − CD34 + cells isolated from BM samples of 2 HDs (n=895) and 5 CMML patients (n=3,161). Each dot represents one cell. Different colors represent the cluster cell type identity (left) or sample origin (right). HSC, hematopoietic stem cells; eMyHPC, early myeloid progenitor cells; dMyHPC, differentiated myeloid progenitors; Ery/MkHPC, erythroid/megakaryocyte hematopoietic progenitor cells. Dashed lines indicate single clusters in each cell type population. (B) Distribution of HD (top) and CMML (bottom) Lin − CD34 + cell types among the clusters shown in . (C) Pathway enrichment analysis of the genes that were significantly upregulated in HSCs (left), eMyHPCs (middle), and dMyHPCs (right) from CMML samples compared with those from HD samples (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown. (D) UMAP of scRNA-seq data for pooled single MNCs isolated from BM samples of 3 HDs (n=9,896) and 5 CMML patients (n=9,319). Each dot represents one cell. Different colors represent the cluster cell type identity (left) or sample origin (right). HSC, hematopoietic stem cells; MKP, megakaryocyte precursors; MyHPC, myeloid hematopoietic progenitor cells; Mono, monocytes; cDC, classical dendritic cells; pDC, plasmacytoid dendritic cells; Prog B, progenitor B-cells; PC, plasma cells; Pre-Ery, pre-erythrocytes; Ery-E, early erythroid precursors; Ery-L, late erythroid precursors; nCD4T, naïve CD4 + T cells; nmCD4T, naïve and memory CD4 + T cells; mCD4T, memory CD4 + T cells; nCD8T, naïve CD8 + T cells; nmCD8T, naïve and memory CD8 + T cells; eCD8T, effector CD8 + T cells; NKC, natural killer cells. Dashed lines indicate single clusters in each cell type population. (E) Distribution of HD (top) and CMML (bottom) cell types among the clusters shown in . (F) Pathway enrichment analysis of the genes that were significantly upregulated in the CMML monocyte clusters compared with those in the HD monocyte clusters shown in (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown.

Article Snippet: For cell sorting applications, MNCs were enriched in CD34 + cells using magnetic-activated cell sorting (MACS) with the CD34 Microbead Kit (catalog number #130-046-702, Miltenyi Biotec, Germany) and further purified by fluorescence-activated cell sorting (FACS) as described below.

Techniques: Isolation, Clinical Proteomics

Journal: bioRxiv

Article Title: Targeting MCL1-driven anti-apoptotic pathways to overcome hypomethylating agent resistance in RAS -mutated chronic myelomonocytic leukemia

doi: 10.1101/2023.04.07.535928

Figure Lengend Snippet: (A) UMAP of scRNA-seq data for pooled single Lin − CD34 + cells isolated from BM samples of 5 CMML patients at diagnosis (n=1,840) and at BP after HMA therapy failure (n=1,711). Each dot represents one cell. Different colors represent the cluster cell type identity (left) or sample origin (right). HSC, hematopoietic stem cells; eMyHPC, early myeloid hematopoietic progenitor cells; dMyHPC, differentiated myeloid hematopoietic progenitor cells; Ery/MkHPC, erythroid/megakaryocyte hematopoietic progenitor cells. Dashed lines indicate single clusters in each cell type population. (B) Distribution of Lin − CD34 + cell types at diagnosis (top) and BP (bottom) among the clusters shown in . (C) Pathway enrichment analysis of the genes that were significantly upregulated in HSCs (left) and dMyHPCs (right) at the time of BP after HMA therapy failure compared with those at diagnosis (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown. (D) UMAP of scATAC-seq data for pooled Lin − CD34 + cells isolated from BM samples obtained from a CMML patient at diagnosis (n=2,027) and at BP after HMA therapy failure (n=2,895). Each dot represents one cell. Different colors represent the cluster identity (left) or sample of origin (right). HSC, hematopoietic stem cells; MyHPC, myeloid progenitor cells; Ery/MkHPC, erythroid/megakaryocyte hematopoietic progenitor cells. (E) Pathway enrichment analysis of genes whose distal elements were enriched in open chromatin regions in HSCs (cluster 1, shown in ) at the time of BP as compared with those at diagnosis (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown.

Article Snippet: For cell sorting applications, MNCs were enriched in CD34 + cells using magnetic-activated cell sorting (MACS) with the CD34 Microbead Kit (catalog number #130-046-702, Miltenyi Biotec, Germany) and further purified by fluorescence-activated cell sorting (FACS) as described below.

Techniques: Isolation, Biomarker Discovery

Journal: bioRxiv

Article Title: Targeting MCL1-driven anti-apoptotic pathways to overcome hypomethylating agent resistance in RAS -mutated chronic myelomonocytic leukemia

doi: 10.1101/2023.04.07.535928

Figure Lengend Snippet: (A) UMAP of scRNA-seq data for pooled single MNCs isolated from BM samples of 6 CMML patients at diagnosis (n=16,372) and at BP after HMA therapy failure (n=19,541). Each dot represents one cell. Different colors represent the cluster cell type identity (left) or sample of origin (right). MyHPC, myeloid hematopoietic progenitors; My/MoP, myelo/monocytic progenitors; Mono, monocytes; cDC, classical dendritic cells; pDC, plasmacytoid dendritic cells; MKP, megakaryocyte precursors; Ery-E, early erythroid precursors; Ery-L, late erythroid precursors; B-cell, B lymphocytes; PC, plasma cells; nCD4T, naïve CD4 + T cells; mCD4T, memory CD4 + T-cells; eCD8T, effector CD8 T-cells, NKC, natural killer cells. Dashed lines indicate single clusters in each cell type population. (B) Distribution of MNC types at diagnosis (top) and (bottom) among the clusters shown in . (C) Pathway enrichment analysis of the genes that were significantly upregulated in the monocytic populations shown in the time of BP after HMA therapy failure compared with those at the time of diagnosis (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown. (D) Numbers of live Lin − CD34 + CD38 − HSCs and Lin − CD34 + CD38 + MyHPCs from CMML patients with BP after treatment with vehicle or 20 nM AMG-176 (n=4) for 48 h. Lines represent means ± SDs. Statistical significance was calculated using a two-tailed Student’s t- test (*** P <0.001; **** P <0.0001). (E) UMAP of scRNA-seq data for pooled single MNCs isolated from BM samples obtained from a representative CMML patient at the time of BP after HMA therapy failure (n=6,209) and after the failure of venetoclax-based therapy (n=6,795). Each dot represents one cell. Different colors represent the cluster cell type identity (left) or the sample of origin (right). HSC, hematopoietic stem cells; MyHPC, myeloid hematopoietic progenitor cells; My/MoP, myelo/monocytic progenitors; Mono, monocytes; Ery/MkHPC, erythroid/megakaryocytic hematopoietic progenitor cells; Ery-E, early erythroid precursors; Ery-L, late erythroid precursors; Pre-E, pre-erythrocytes; mCD8T, memory CD8 + T cells; eCD8T, effector CD8 + T cells; NKC, natural killer cells. (F) Pathway enrichment analysis of the genes that were significantly upregulated in MyHPCs at the time of venetoclax failure compared with those at the time of HMA therapy failure (adjusted P ≤ 0.05). The top 10 Hallmark gene sets are shown. (G) Distribution of myeloid cell types among the myeloid compartments at HMA therapy (top) and venetoclax-based therapy (bottom) failure. (H) Proposed working model of RAS pathway–mutated CMML initiation and progression after HMA and venetoclax-based therapies. Compared with physiological adult hematopoiesis (top left), RAS pathway–mutated CMML HSPCs undergo proliferation and monocytic differentiation in response to inflammatory responses while maintaining an intact apoptotic program. Inflammatory reprograming is exacerbated in downstream monocytic populations, which contributes to disease maintenance (bottom left). At BP after HMA therapy failure, RAS pathway–mutated CMML HSCs undergo epigenetic reprogramming and drive the expansion of downstream MyHPCs. MyHPCs and downstream monocytes rely on NF- K B signaling–mediated anti-apoptotic pathways to maintain survival and suppress the immune microenvironment (bottom right). NF- K B signaling–mediated survival pathway activation persists after venetoclax therapy and leads to treatment resistance and failure (top right).

Article Snippet: For cell sorting applications, MNCs were enriched in CD34 + cells using magnetic-activated cell sorting (MACS) with the CD34 Microbead Kit (catalog number #130-046-702, Miltenyi Biotec, Germany) and further purified by fluorescence-activated cell sorting (FACS) as described below.

Techniques: Isolation, Biomarker Discovery, Clinical Proteomics, Two Tailed Test, Activation Assay

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: CD34+ cells from three SCD patient donors were electroporated with ABE8e-NRCH mRNA or RNP using an sgRNA targeting the SCD mutant HBB codon. (a) The edited region of HBB with the target A at protospacer position 7 shown in blue along with potential bystander edits in green (silent), brown (silent), and red (non-silent). (b) Editing efficiencies by HTS at target and bystander adenines, and indels after 6 days in stem-cell culture media following electroporation. (c) Proportion of β-like globin proteins by HPLC of reticulocyte lysates after 18 days in differentiation media following electroporation. (d) Representative phase-contrast images of reticulocytes derived from unedited or edited donor HSPCs incubated 8 hours in 2% O2. Nine images of >50 cells each were collected per sample. Scale bar=50 μm. (e) Quantification of sickled reticulocytes from counting >300 randomly selected cells by a blinded observer from images as in (d). (f) Venn diagram showing candidate off-target sites nominated by Cas-OFFinder and CIRCLE-seq, and nominated sites for which off-target editing was observed by targeted DNA sequencing in SCD patient CD34+ cells electroporated with ABE8e-NRCH mRNA. (g) Predicted genomic features of validated off-target sites. TTS, ≤1 kb from the transcription termination site; UTR, untranslated region. (h) ABE8e-NRCH-treated HSPCs from two different SCD patient donors were sequenced at 697 potential off-target sites. The histogram shows the number of validated off-target base editing sites binned by average percentage of sequencing reads for each site with any A•T-to-G•C mutations in protospacer nucleotides 4-10. Bar values in (b), (c), and (e) and error bars reflect mean±SD of three independent biological replicates, with individual values shown as dots.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Mutagenesis, Stem Cell Culture, Electroporation, Derivative Assay, Incubation, DNA Sequencing, Sequencing

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: Representative, immuno-flow cytometry for erythroid maturation stage markers42,43 at culture days 7 and 14. Top: gating strategy to identify single cells expressing the erythroid marker hCD235a. Bottom: gating strategy to track the progress of erythroid maturation based on expression of CD49D and Band3 in hCD235a+ cells. SSC-A: Side scatter area. SSC-W: Side scatter width. FSC-A: Forward scatter area.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Flow Cytometry, Expressing, Marker

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: Reverse-phase HPLC chromatograms of erythroid cell lysates at culture day 18, with β-like globins and their associated fractions marked near the associated peak. Data from the most efficiently edited donor is shown. Red arrows indicate the start and end of globin chain peaks.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: High Performance Liquid Chromatography, Derivative Assay, In Vitro

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: CIRCLE-seq read counts obtained for each verified off-target site and the alignment of each site to the guide sequence are shown. Bar graphs show the percentage of sequencing reads containing A•T-to-G•C mutations within protospacer positions 4-10 at on-and off-target sites in genomic DNA samples from patient CD34+ HSPCs treated with ABE8e-NRCH mRNA, protein, or untreated controls (n=4). Note that the mutation frequency shown is summed across all reads with one or more A•T-to-G•C mutations in this window. Sequencing errors therefore accumulate in control samples compared to standard sequencing error frequencies for a single nucleotide. Bar values and error bars reflect mean±SD.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Sequencing, Mutagenesis, Control

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: Bar graph showing the percentage of sequencing reads containing alleles harboring indels at on-and off-target sites in genomic DNA samples from patient CD34+ HSPCs treated with ABE8e-NRCH mRNA, protein, or untreated controls (n=4). Bar values and error bars reflect mean±SD.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Sequencing

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: CD34+ HSPCs from three HBBS/S SCD patient donors were electroporated with ABE8e-NRCH mRNA and sgRNA targeting the SCD mutant HBB codon. 2-5x105 treated cells were transplanted into NBSGW mice via tail-vein injection. Mice were analyzed 16 weeks after transplantation. (a) Experimental workflow. (b) Engraftment measured by percentage of human CD45+ (hCD45+) cells in recipient mouse bone marrow. (c) Human B-cells (hCD19+), myeloid cells (hCD33+), and T-cells (hCD3+) cells in recipient mouse bone marrow shown as percentages of the hCD45+ population. (d) Human erythroid precursors (hCD235a+) in recipient mouse bone marrow shown as percentage of human and mouse CD45− cells, (e) HBBS-to-HBBG editing efficiencies in human donor CD34+ cell-derived lineages from recipient bone marrow. Erythroid, myeloid, B-cell, and HSPC human lineages were collected using antibodies that recognize hCD235a, hCD33, hCD19, and hCD34, respectively, (f) Clonal editing outcomes determined by single-cell 5’ RNA-seq in CD235a+ cells from the bone marrow of two edited mice. (g) Proportions of β-like globin proteins by HPLC of human donor-derived reticulocytes isolated from recipient mouse bone marrow. (h) Representative phase-contrast images of human reticulocytes from bone marrow incubated 8 hours in 2% O2. Nine images of >50 cells each were collected per sample. Scale bar=50 μm. (i) Quantification of sickled cells as in Fig. 1e. n=14 mice receiving edited cells and n=13 mice receiving unedited cells in b-e, g, and i. Triangle, square, and circle symbols represent HSPCs from three different SCD donors. Plotted values and error bars reflect mean±SD. Statistical significance was assessed by one-way ANOVA in i and by two-tailed Student’s t-test elsewhere; “ns”, not significant.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Mutagenesis, Injection, Transplantation Assay, Derivative Assay, RNA Sequencing, Isolation, Incubation, Two Tailed Test

Journal: Nature

Article Title: Base editing of hematopoietic stem cells rescues sickle cell disease in mice

doi: 10.1038/s41586-021-03609-w

Figure Lengend Snippet: CD34+ HSPCs from three HBBS/S SCD patient donors were electroporated with ABE8e-NRCH RNP using a single guide RNA (sgRNA) targeting the SCD mutant codon, followed by transplantation of 2-5x105 treated cells into NBSGW mice via tail-vein injection. Mice were sacrificed and analyzed 16 weeks after transplantation, (a) Experimental workflow, (b) Engraftment measured by the percentage of human donor CD45+ cells (hCD45+ cells) in recipient mouse bone marrow, (c) Human B-cells (hCD19+), myeloid cells (hCD33+), and T-cells (hCD3+) cells in recipient mouse bone marrow, shown as percentages of the total hCD45+ population. (d) Human erythroid precursors (hCD235a+) in recipient mouse bone marrow shown as percentage of total human and mouse CD45−cells. (e) On-target (A7, Fig. 1a) editing efficiencies in human donor CD34+ cell-derived lineages purified from recipient bone marrow by fluorescence-activated cell sorting. Erythroid, myeloid, B-cell, and HSPC human lineages were collected using antibodies that recognize hCD235a, hCD33, hCD19, and hCD34+, respectively. Statistical significance was assessed by one-way ANOVA to compare groups; “ns”, not significant. (f) Percentages of β-like globin proteins determined by reverse-phase HPLC analysis of human donor-derived reticulocytes isolated from recipient mouse bone marrow. (g) Representative phase contrast images of human reticulocytes purified from bone marrow and incubated for 8 hours in 2% O2. Nine images of >50 cells per image were collected per sample. Scale bar=50 μm. (h) Quantification of sickled cells calculated by counting images after incubation for 8 hours in 2% O2 such as in (g). More than 300 randomly selected cells per sample were counted by a blinded observer. n=14 total mice analyzed in panels b-f; triangle, square, and circle symbols represent samples from three different SCD CD34+ HSPC donors. Negative control data is shared with Figure 2. Bar values and error bars reflect mean±SD. Statistical significance between treated and untreated samples was assessed by a two-tailed Student’s t-test; “ns”, not significant.

Article Snippet: CD34 + HSPCs or CD235a + erythroblasts were isolated with magnetic beads, using the human-specific CD34 MicroBead Kit UltraPure (Miltenyi Biotec Inc., catalog # 130-100-453) and CD235a (glycophorin A) MicroBeads, human, (Miltenyi Biotec Inc., catalog # 130-050-501).

Techniques: Mutagenesis, Transplantation Assay, Injection, Derivative Assay, Purification, Fluorescence, FACS, Isolation, Incubation, Negative Control, Two Tailed Test

Journal: Stem Cells Translational Medicine

Article Title: Transient Inhibition of the JNK Pathway Promotes Human Hematopoietic Stem Cell Quiescence and Engraftment

doi: 10.1093/stcltm/szac019

Figure Lengend Snippet: Transient inhibition of the JNK pathway increased the HSC number in CB CD34 + cells. ( A ) Schematic of the experimental design. STF represents basic culture medium (StemSpan SFEM II supplemented with 100 ng/mL SCF, 50 ng/mL TPO, and 100 ng/mL Flt3L). Conditions that increased the frequency of Lin - CD34 + CD45RA − cells compared with culturing in basic culture medium were regarded as positive hits. ( B ) Bar plot showing the percentage of Lin − CD34 + CD45RA − cells in CB CD34 + cells cultured in medium supplemented with cytokines only (Ctrl), DMSO, AEG3482, SP600125, or JNK-IN-8 for 24 h. ( n = 3) ( C ) Representative FACS plots showing the expression of the indicated surface markers on DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells. ( D and E ) Bar plot showing the frequencies of Lin − CD34 + CD45RA − ( D ) and Lin − CD34 + CD38 − CD45RA − ( E ) cells in DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells. ( F ) Bar plot showing the Lin − CD34 + CD38 − CD45RA − cell count in 2 × 10 4 DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells. ( G ) The frequency of engrafted human CD45 + cells in the PB of recipient mice receiving DMSO-treated, uncultured, or JNK-IN-8-treated CB CD34 + cells measured at 4-, 8-, and 12-weeks post-transplantation. ( H ) HSC frequencies presented as 1/uncultured CD34 + cell equivalent for DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells calculated with ELDA software at 8 weeks post-transplantation. The required CI was 95%. The cutoff for positive engraftment was set as more than 0.01% human CD45 + cells in the PB of the recipient ( n = 5 mice for each group). ( I ) Number of repopulating HSCs per 1 × 10 5 CD34 + cells in the DMSO-treated, uncultured, and JNK-IN-8-treated groups ( n = 5 mice for each group). See also ; . All data are shown as the mean value ± SD. Statistical significance was assessed using one-way ANOVA if not mentioned. ns, not significant; * P <.05; ** P < .01; *** P < .001.

Article Snippet: Human CB CD34 + cells were isolated with a CD34 MicroBead Kit (Miltenyi Biotec) according to the manufacturer’s instructions and cultured in StemSpan SFEMII (Stem Cell Technologies) supplemented with recombinant human SCF (100 ng/mL, StemImmune LLC), recombinant human FLT3L (100 ng/mL, StemImmune LLC), recombinant human TPO (50 ng/mL, StemImmune LLC), and the indicated molecules.

Techniques: Inhibition, Cell Culture, Expressing, Cell Counting, Transplantation Assay, Software

Journal: Stem Cells Translational Medicine

Article Title: Transient Inhibition of the JNK Pathway Promotes Human Hematopoietic Stem Cell Quiescence and Engraftment

doi: 10.1093/stcltm/szac019

Figure Lengend Snippet: Transient inhibition of the JNK pathway increased the LT-HSC number during CB CD34 + cell isolation. ( A ) The frequency of engrafted human CD45 + cells in the PB of recipient mice receiving conventionally isolated (Ctrl) or JNK-IN-8-treated CB CD34 + cells measured at 4-, 8-, 12-, and 16-weeks post-transplantation. ( B – D ) Representative FACS plots showing human CD45 + cell engraftment in the PB ( B ), BM ( C ), and spleen ( D ) of recipient mice at 20 weeks after transplantation of conventionally isolated (Ctrl) or JNK-IN-8-treated CB CD34 + cells. ( E ) HSC frequencies presented as 1/uncultured CD34 + cell equivalent for conventionally isolated (Ctrl) and JNK-IN-8-treated CB CD34 + cells calculated with ELDA software at 20 weeks post-transplantation. The required CI was 95%. The cutoff for positive engraftment was set as more than 0.1% human CD45 + cells in the BM of the recipient ( n = 15 mice for each group with 3 independent experiments, **** P < .0001). ( F ) Number of LT-HSCs per 1 × 10 5 cells in conventionally isolated (Ctrl) and JNK-IN-8-treated CB CD34 + cells. See also and .( G ) Representative FACS plots showing human B (CD19 + ) and myeloid cell (CD33/CD14/CD11b + ) repopulation in recipient BM (gated in hCD45 + ) at 20 weeks post-transplantation. ( H ) Bar plot showing the lineage distribution of engrafted human CD45 + cells in recipient BM. B, CD19 + B cells; M, CD33 + /CD14 + /CD11b + myeloid cells; n = 5. ( I ) Representative FACS plots showing human T-cell (hCD45 + CD3 + ) repopulation in the recipient thymus at 20 weeks post-transplantation. ( J ) Level of human CD45 + cell engraftment in the PB of 2° recipients at the indicated doses of BM cells from primary recipients at 14 weeks post-transplantation. ( K ) 2° SRC frequencies in the BM of the primary recipients receiving conventionally isolated (Ctrl) and JNK-IN-8-treated CB CD34 + cells calculated with ELDA software at 14 weeks post-transplantation. The required CI was 95%. The cutoff for positive engraftment was set as more than 0.01% human CD45 + cells in the PB of the recipient ( n = 5 mice for each group, *** P < .001). ( L ) Number of 2° SRC per 1 × 10 7 cells in conventionally isolated (Ctrl) and JNK-IN-8-treated CB CD34 + cells. See also and . All data are shown as the mean value ± SD. Statistical significance was assessed using one-way ANOVA if not mentioned. ns, not significant; * P < .05; ** P < .01; *** P < .001; **** P < .0001.

Article Snippet: Human CB CD34 + cells were isolated with a CD34 MicroBead Kit (Miltenyi Biotec) according to the manufacturer’s instructions and cultured in StemSpan SFEMII (Stem Cell Technologies) supplemented with recombinant human SCF (100 ng/mL, StemImmune LLC), recombinant human FLT3L (100 ng/mL, StemImmune LLC), recombinant human TPO (50 ng/mL, StemImmune LLC), and the indicated molecules.

Techniques: Inhibition, Cell Isolation, Isolation, Transplantation Assay, Software

Journal: Stem Cells Translational Medicine

Article Title: Transient Inhibition of the JNK Pathway Promotes Human Hematopoietic Stem Cell Quiescence and Engraftment

doi: 10.1093/stcltm/szac019

Figure Lengend Snippet: Transient inhibition of the JNK pathway promoted HSC quiescence, preventing HSCs from undergoing cell cycle entry and metabolic activation. ( A ) Heatmap showing gene expression in DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells. ( B ) Expression by RNA-seq of the indicated genes in DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells. The fpkm value for uncultured cells is normalized to 1.0; n = 2. ( C and D ) GSEA plots showing enrichment of quiescent-HSC-enriched ( C ) and 120 HSC-associated ( D ) gene sets in the indicated groups. DMSO, DMSO-treated CB CD34 + cells; uncultured, uncultured CB CD34 + cells; JNK-IN-8, JNK-IN-8-treated CB CD34 + cells. Each group contained 2 replicates. ( E ) Bar plot showing the cell cycle status of DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells ( n = 3). ( F and G ) Representative FACS plots ( F ) and bar plot ( G ) showing glucose uptake activity (indicated by the fluorescence intensity of 2-NBDG) of DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells ( n = 3). ( H and I ) Representative FACS plots ( H ) and bar plot ( I ) showing ROS levels (indicated by the fluorescence intensity of DCFDA) of DMSO-treated, uncultured, and JNK-IN-8-treated CB CD34 + cells ( n = 3). ( J ) Schematic of the proposed model demonstrating how transient JNK inhibition regulates CB HSPC engraftment through its roles in quiescence and stemness. All data are shown as the mean value ± SD. Statistical significance was assessed using one-way ANOVA if not mentioned. ns, not significant; ** P <.01; *** P < .001; **** P < .0001.

Article Snippet: Human CB CD34 + cells were isolated with a CD34 MicroBead Kit (Miltenyi Biotec) according to the manufacturer’s instructions and cultured in StemSpan SFEMII (Stem Cell Technologies) supplemented with recombinant human SCF (100 ng/mL, StemImmune LLC), recombinant human FLT3L (100 ng/mL, StemImmune LLC), recombinant human TPO (50 ng/mL, StemImmune LLC), and the indicated molecules.

Techniques: Inhibition, Activation Assay, Gene Expression, Expressing, RNA Sequencing, Activity Assay, Fluorescence

Journal: Pharmaceutics

Article Title: Exosome-Modified Liposomes Targeted Delivery of Thalidomide to Regulate Treg Cells for Antitumor Immunotherapy

doi: 10.3390/pharmaceutics15041074

Figure Lengend Snippet: Thalidomide and its analogs inhibit Treg expansion mediated by TNF in vitro. MACS was used to purify CD4 + T cells from lymphocytes. CD4 + T cells were stimulated in the presence of IL-2 (10 ng/mL), with or without TNF (20 ng/mL), for 72 h. Thalidomide and its analogs with two concentrations, 50 μM and 100 μM, with or without TNF (20 ng/mL), for 72 h. Flow cytometry was used to analyze proportion of Foxp3 + Tregs. ( A ) Typical flow cytometry data of CD4 + Foxp3 + Treg cells proportion. ( B – D ) Summarized data of CD4 + Foxp3 + Treg cells proportion. Data (means ± SEM, n = 9), pooled from 2 or 3 separate experiments with similar results. * p < 0.05, as compared with IL-2 alone group. # p < 0.05, ## p < 0.01, as compared with IL-2 plus TNF group.

Article Snippet: CD4 (L3T4) MicroBeads mouse isolation kit (#130-097-054) were purchased from Miltenyl (Bergisch Gladbach, Germany).

Techniques: In Vitro, Flow Cytometry

Journal: Pharmaceutics

Article Title: Exosome-Modified Liposomes Targeted Delivery of Thalidomide to Regulate Treg Cells for Antitumor Immunotherapy

doi: 10.3390/pharmaceutics15041074

Figure Lengend Snippet: Thalidomide and its analogs inhibit Treg proliferative expansion mediated by TNF in vitro. CFSE was used to label CD4 + T cells purified by MACS from lymphocytes. CFSE-labeled CD4 + T cells were stimulated in the presence of IL-2 (10 ng/mL), with or without TNF (20 ng/mL), for 72 h. Thalidomide and its analogs with two concentrations, 50 μM and 100 μM, were added. Flow cytometry was used to analyze Treg proliferation by gating on CD4 + Foxp3 + cells. ( A ) Typical flow cytometry data of CD4 + Foxp3 + Treg cells proliferation. ( B – D ) Summarized data of CD4 + Foxp3 + Treg cells proliferation. Data (means ± SEM, n = 9), pooled from 2 or 3 separate experiments with similar results. *** p < 0.001, as compared with IL-2 alone group. # p < 0.05; ## p < 0.01, as compared with IL-2 plus TNF group.

Article Snippet: CD4 (L3T4) MicroBeads mouse isolation kit (#130-097-054) were purchased from Miltenyl (Bergisch Gladbach, Germany).

Techniques: In Vitro, Purification, Labeling, Flow Cytometry

Journal: Pharmaceutics

Article Title: Exosome-Modified Liposomes Targeted Delivery of Thalidomide to Regulate Treg Cells for Antitumor Immunotherapy

doi: 10.3390/pharmaceutics15041074

Figure Lengend Snippet: HE-THD inhibits Treg expansion and proliferation mediated by TNF in vitro. CellTrace violet-labeling lymphocyte were stimulated in the presence of IL-2 (10 ng/mL), with or without TNF (20 ng/mL) for 72 h. HE-THD was added with concentration of 50 μM. Flow cytometry was used to analyze proportion and proliferation of Foxp3 + Tregs. ( A ) Typical Flow cytometry data of CD4 + Foxp3 + Treg cells proportion. ( B , D ) Summarized data of CD4 + Foxp3 + Treg cells proportion. ( C ) Typical flow cytometry data of CD4 + Foxp3 + Treg cells proliferation. Data (means ± SEM, n = 9), pooled from 2 or 3 separate experiments with similar results. ** p < 0.01, *** p < 0.001, as compared with IL-2 plus TNF group.

Article Snippet: CD4 (L3T4) MicroBeads mouse isolation kit (#130-097-054) were purchased from Miltenyl (Bergisch Gladbach, Germany).

Techniques: In Vitro, Labeling, Concentration Assay, Flow Cytometry

Journal: Pharmaceutics

Article Title: Exosome-Modified Liposomes Targeted Delivery of Thalidomide to Regulate Treg Cells for Antitumor Immunotherapy

doi: 10.3390/pharmaceutics15041074

Figure Lengend Snippet: Upregulation of TNFR2 expression on Tregs induced by TNF is abrogated by HE-THD. Lymphocytes were cultured in the presence of IL-2 (10 ng/mL), or IL-2 + TNF (20 ng/mL), with medium alone or with HE-THD (50 μM). The cells were cultured for 72 h. The proportion of surface expression of TNFR2 in CD4 + Foxp3 + Tregs was analyzed with FACS. ( A ) Typical flow cytometry data of surface TNFR2 expression on CD4 + Foxp3 + Treg cells. ( B ) Summarized data of proportion of TNFR2 on CD4 + Foxp3 + Treg cells. ( C ) Mean fluorescence intensity (MFI) for TNFR2 expression. Representative FACS data from at least three separate experiments with similar results are shown on the upper panel. Summarized data (mean ±SEM), pooled from 3 to 4 separate experiments ( n = 9~12). * p < 0.05, ** p < 0.01, as compared with “TNF + IL-2” group (without HE-THD).

Article Snippet: CD4 (L3T4) MicroBeads mouse isolation kit (#130-097-054) were purchased from Miltenyl (Bergisch Gladbach, Germany).

Techniques: Expressing, Cell Culture, Flow Cytometry, Fluorescence

Journal: The Journal of Immunology Author Choice

Article Title: Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4 + CD25 + FOXP3 + T Cells

doi: 10.4049/jimmunol.2300019

Figure Lengend Snippet: FOXP3 expression in CD4+CD25+ T cells induced by optimal and suboptimal stimulation with SEC1 in a Vβ-specific way. Human PBMCs depleted of CD25+ cells were stimulated with SEC1 at concentrations inducing optimal (1 µg/ml) or suboptimal (1 ng/ml) stimulation for up to 8 d. FOXP3 expression before and during stimulation was measured using flow cytometry. (A) The percentage (mean ± SEM) of CD4+CD25+FOXP3+ T cells was obtained with data combined from three donors. Data shown are a single representative of three donors and gated on live/CD4+ T cells. (B) Expression of Vβ2 and Vβ14, representing nonspecific and specific Vβ subsets to SEC1, respectively, was measured by flow cytometry before and after stimulation with SEC1 (1 ng/ml) for 6 d. The percentage of Vβ-positive population (dark gray, upper histogram) was measured on the basis of fluorescence minus one control (light gray, lower histogram) in CD4+ T cells. (C) The percentage of CD25+FOXP3+ cells in Vβ positive population was measured after suboptimal stimulation for 6 d. Data shown are combined from three independent experiments (n = 9), and statistical difference was analyzed using the Student t test (**p < 0.001).

Article Snippet: CD4 + CD25 + T cells were purified from optimal (day 4) and suboptimal (day 6) stimulation of PBMCs with SEC1 by negative selection using a human CD4 + isolation kit (Miltenyi Biotec), followed by a human CD25 microbeads II kit (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Expressing, Flow Cytometry, Fluorescence, Control

Journal: The Journal of Immunology Author Choice

Article Title: Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4 + CD25 + FOXP3 + T Cells

doi: 10.4049/jimmunol.2300019

Figure Lengend Snippet: CD4+CD25+ T cells induced by suboptimal stimulation are immunosuppressive predominantly mediated by cell-to-cell contact and partly by soluble factors. (A and B) For the MLR assay, CFSE-stained naive CD4+CD25− T cells were stimulated with anti-CD3/CD28 beads alone or cocultured with CD4+CD25+ T cells induced by suboptimal (1 ng/ml) or optimal (1 µg/ml) stimulation stained with CellTrace FarRed dye for 4 d. (B) Percentage (mean ± SEM) of nonproliferating responder cells from three independent experiments (n = 9). (C and D) CD4+CD25+ T cells induced by suboptimal stimulation with SEC1 (1 ng/ml) were stained with CellTrace FarRed and treated with mitomycin C (indicated as M) for the contact-dependent suppression assay or placed in the top chamber of a Transwell (indicated as T) for the soluble factor–mediated suppression assay. CFSE-labeled naive CD4+CD25− T cells were cocultured as responder cells and stimulated with anti-CD3/CD28 beads. (D) The percentage (mean ± SEM) of nonproliferating CD4+ T cells was obtained by combining results from three independent experiments (n = 9). (E and F) Neutralizing mAb (10 µg/ml) for galectin-1 (indicated as G) was added in an MLR. In some experiments, CD4+CD25+ T cells were treated with mitomycin C (indicated as M) or placed in the top chamber of the Transwell (indicated as T). (F) The percentage (mean ± SEM) of nonproliferating CD4+ T cells was obtained from combined results from three independent experiments. Statistical difference was determined by one-way ANOVA followed by Tukey honestly significant difference test (**p < 0.01, ***p < 0.001).

Article Snippet: CD4 + CD25 + T cells were purified from optimal (day 4) and suboptimal (day 6) stimulation of PBMCs with SEC1 by negative selection using a human CD4 + isolation kit (Miltenyi Biotec), followed by a human CD25 microbeads II kit (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Mlr Assay, Staining, Suppression Assay, Labeling

Journal: The Journal of Immunology Author Choice

Article Title: Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4 + CD25 + FOXP3 + T Cells

doi: 10.4049/jimmunol.2300019

Figure Lengend Snippet: Differential activation of PI3K-Akt-mTOR signaling pathway by optimal and suboptimal stimulation with SEC1. PBMCs depleted of CD25+ T cells were stimulated with optimal or suboptimal concentrations of SEC1 for up to 6 d, and total protein was extracted at the indicated time points. Immunoblot analysis of total and/or phosphorylated S6, Akt, mTOR, PTEN, and β-actin was performed. (A) Representative immunoblot data are shown, and band intensity was measured using ImageJ software. (B) Relative band intensity was calculated by normalizing to its unphosphorylated form (for S6 and Akt) or β-actin (for mTOR and PTEN). Results were combined from two independent experiments. (C) PTEN mRNA expression was measured by quantitative PCR, analysis and relative expression to day 0 was calculated after normalization to β-actin. Statistical significance between optimal and suboptimal stimulation was determined by two-way ANOVA with Tukey honestly significant difference test (*p < 0.05, **p < 0.01, ***p < 0.001).

Article Snippet: CD4 + CD25 + T cells were purified from optimal (day 4) and suboptimal (day 6) stimulation of PBMCs with SEC1 by negative selection using a human CD4 + isolation kit (Miltenyi Biotec), followed by a human CD25 microbeads II kit (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Activation Assay, Western Blot, Software, Expressing, Real-time Polymerase Chain Reaction

Journal: The Journal of Immunology Author Choice

Article Title: Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4 + CD25 + FOXP3 + T Cells

doi: 10.4049/jimmunol.2300019

Figure Lengend Snippet: Differential subcellular localization of FOXP3 isoforms by optimal and suboptimal stimulation with SEC1. (A) Two anti-FOXP3 Ab clones were used to detect FOXP3 isoforms. For (B)–(D), PBMCs depleted of CD25+ cells were stimulated with optimal (4 d; Op) or suboptimal (6 d; Sub) stimulation with SEC1. (B) Confocal microscopic images were taken after staining with anti-FOXP3 Ab conjugated with Alexa Fluor 488 (green). Nucleus was counterstained with DAPI (blue). Original magnification ×40. (C) Cell lysates were fractionated and blotted against anti-FOXP3 Abs and control markers (Erk1/2 for cytoplasmic fraction, histone H3 for nucleic fraction). Cell lysates from day 0 (unstimulated; Un) were used as a negative control. (D) Quantification of Western blot bands was performed using ImageJ software. The bar graph represents the relative expression levels of FOXP3 proteins normalized to unstimulated sample. Results were combined from two independent experiments (**p < 0.01, ***p < 0.001).

Article Snippet: CD4 + CD25 + T cells were purified from optimal (day 4) and suboptimal (day 6) stimulation of PBMCs with SEC1 by negative selection using a human CD4 + isolation kit (Miltenyi Biotec), followed by a human CD25 microbeads II kit (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Clone Assay, Staining, Control, Negative Control, Western Blot, Software, Expressing

Journal: The Journal of Immunology Author Choice

Article Title: Stimulation Strength Determined by Superantigen Dose Controls Subcellular Localization of FOXP3 Isoforms and Suppressive Function of CD4 + CD25 + FOXP3 + T Cells

doi: 10.4049/jimmunol.2300019

Figure Lengend Snippet: Blockade of PI3K signaling induced suppressive functionality by optimal stimulation with SEC1. PBMCs depleted of CD25+ T cells were stimulated with optimal stimulation concentration of SEC1 (1 µg/ml) in the presence of vehicle (DMSO; Veh) or PI3K inhibitor (LY294002, 10 µM; PI3Ki) for 4 d. (A) Expression of CD4+CD25+FOXP3+ T cells was analyzed using flow cytometry. A representative flow plot gated in live/CD4+ T cells (left) and the percentage (mean ± SEM) of CD4+CD25+FOXP3+ T cells (right). (B) Percentage of CD25+FOXP3+ cells in Vβ14+ T cells. Data shown are combined from three independent experiments (n = 9), and statistical difference was analyzed using the Student t test (*p < 0.05). (C) After 4 d of stimulation, CD4+CD25+ T cells were isolated, labeled with CellTrace FarRed dye, and cocultured with CFSE-labeled naive CD4+CD25− T cells isolated from same donor. Anti-CD3/CD28 beads were added as a stimulant, and dilution of CFSE signal was measured on day 4 after coculture using flow cytometry. Single representative flow data are shown (left), and the percentage (mean ± SEM) of nonproliferating CD4+ T cells was obtained by combining results from two independent experiments (n = 5) (right). Statistical significance was determined by ANOVA followed by Tukey honestly significant difference test (*p < 0.05, ***p < 0.001). (D) Confocal microscopic images were taken after staining with anti-FOXP3 Ab conjugated with Alexa Fluor 488 (green). The nucleus was counterstained with DAPI (blue). Original magnification ×40.

Article Snippet: CD4 + CD25 + T cells were purified from optimal (day 4) and suboptimal (day 6) stimulation of PBMCs with SEC1 by negative selection using a human CD4 + isolation kit (Miltenyi Biotec), followed by a human CD25 microbeads II kit (Miltenyi Biotec) according to the manufacturer’s instructions.

Techniques: Concentration Assay, Expressing, Flow Cytometry, Isolation, Labeling, Staining

Journal: Oncoimmunology

Article Title: Bendamustine with total body irradiation conditioning yields tolerant T-cells while preserving T-cell-dependent graft-versus-leukemia

doi: 10.1080/2162402X.2020.1758011

Figure Lengend Snippet: BEN-TBI conditioning improves GvHD independently of donor Tregs. (a) BALB/c recipient mice received 40 mg/kg BEN iv or 200 mg/kg CY ip on day −2, 400 cGy TBI on day −1, and 10 7 BM with 3 × 10 6 SC from naïve C57BL/6 mice on day 0. Peripheral blood was collected on days +7, +14, +21, +35, and +70 and stained for CD4, CD25, and FoxP3 for flow cytometric analysis. CD4+ CD25+ FoxP3+ cells were considered Tregs. Representative flow cytometry histograms from day +35 are shown. CBCs were determined and used to calculate absolute cell numbers. Average absolute numbers of cells per μL of blood are shown with SEM. Pooled data from 4 experiments are shown, n = 19 mice/group. (b) BALB/c recipient mice received 40 mg/kg BEN iv or 200 mg/kg CY ip on day −2, 400 cGy TBI on day −1, and 10 7 BM with 3 × 10 6 SC from naïve C57BL/6 mice on day 0. Splenic Tregs were isolated on day 0 (prior to transplant), +7, and +14. Tregs were plated with anti-CD3/CD28 bead activated, CellTrace Violet-stained T-cells from naïve C57BL/6 mice in a suppression assay. T-cells were plated at 100,000 cells per well in a 96-well U-bottom plate. Tregs were plated at a ratio of 1 Treg to 2 T-cells. Proliferation was assessed by flow after 3 days of co-culture. Average % proliferation with SEM is shown, compared to a control with no Tregs. Pooled data from 2 experiments are shown, n = 4–7 mice/group. (c) BALB/c recipient mice received 40 mg/kg BEN iv or 200 mg/kg CY ip on day −2, 400 cGy TBI on day −1, and 10 7 TCD-BM with 3 × 10 6 tT with or without CD25+ cell depletion from naïve C57BL/6 mice on day 0. Survival is shown. CY+TBI+TCD-BM+tT vs. CY+TBI+TCD-BM+CD25- tT p = .0857 , BEN+TBI+TCD-BM+CD25- tT vs. CY+TBI+TCD-BM+CD25- tT p = .0005 . (d) CD4+ CD62L+ T-cells were isolated from naïve BALB/c mouse spleens and cultured with 5 ng/mL TGF-β and a 1:1 ratio of anti-CD3/CD28 beads for 3 days in the presence of various concentrations of BEN. Cells were phenotyped and the % CD4+ CD25+ FoxP3+ of all cells remaining at the end of the culture was assessed, as well as viability. BEN was washed out and cells were plated in a suppression assay at a ratio of 1 Treg to 1 T-cell (CD3/CD28 activated, CellTrace Violet-stained, from naïve C57BL/6 mice). After 3 days of co-culture, proliferation was assessed by flow cytometry. Representative flow cytometry plots are shown, with proliferation index (PI) indicated, with higher proliferation indices representing greater proliferation. The T-cells + beads plot represents activated T-cells without Tregs (a positive control for proliferation). Averages are shown with SEM. Pooled data from 3 experiments is shown, n = 3/condition.

Article Snippet: CD25+ cells were depleted from tT cells using CD25 Microbeads (Miltenyi Biotec; 130-091-072), resulting in less than 0.4% remaining CD25+ FoxP3+ cells (data not shown).

Techniques: Staining, Flow Cytometry, Isolation, Suppression Assay, Co-Culture Assay, Control, Cell Culture, Positive Control

Journal: Oncoimmunology

Article Title: Bendamustine with total body irradiation conditioning yields tolerant T-cells while preserving T-cell-dependent graft-versus-leukemia

doi: 10.1080/2162402X.2020.1758011

Figure Lengend Snippet: BEN-TBI does not result in appreciable donor T-cell phenotypic differences post-transplant when compared to CY-TBI. (a–c) BALB/c recipient mice received 40 mg/kg BEN iv or 200 mg/kg CY ip on day −2, 400 cGy TBI on day −1, and 10 7 TCD-BM from naïve C57BL/6 mice with 3 × 10 6 CellTrace Violet-stained tT from naïve BoyJ mice on day 0. Blood and spleen were collected on day +3. (a) % donor T-cells (CD45.1+) was determined by flow cytometry. Using CBCs determined by HemaVet analysis, absolute number of donor T-cells was calculated. (b) After gating on CD45.1+ cells (representing donor T-cells), CellTrace Violet dilution was analyzed using ModFit software to determine proliferation index. Representative CellTrace Violet dilution is shown. (c) Within the CD45.1+ gate, cells were stratified by CellTrace high (non- proliferative) and CellTrace low (proliferative) and CD25 and CD47 expression were analyzed by flow cytometry. Pooled data from 2 experiments with line at mean are shown, n = 6–7 mice/group. * p < .05 , ** p < .01 . (d-g) BALB/c recipient mice received 40 mg/kg BEN iv or 200 mg/kg CY ip on day −2, 400 cGy TBI on day −1, and 10 7 BM with 3 × 10 6 SC from naïve C57BL/6 mice on day 0. Peripheral blood was collected on days +7, +14, +21, +35, and +70 and stained for CD8, CD4, Tbet, GATA3, RORγt, CD134, CD278, PD-1, TIM3, CTLA-4, and CD272. CBCs were determined and used to calculate absolute cell numbers. Average absolute numbers of cells per μL of blood are shown with SEM. Representative flow plots from day +7 with fluorescence minus one (FMO) controls are shown (e). (d) Pooled data from 4 experiments are shown, n = 19 mice/group. (e-g) Pooled data from 2 experiments are shown, n = 10 mice/group. ** p < .01.

Article Snippet: CD25+ cells were depleted from tT cells using CD25 Microbeads (Miltenyi Biotec; 130-091-072), resulting in less than 0.4% remaining CD25+ FoxP3+ cells (data not shown).

Techniques: Staining, Flow Cytometry, Software, Expressing, Fluorescence

Journal: International immunopharmacology

Article Title: USP25 deficiency promotes T cell dysfunction and transplant acceptance via mitochondrial dynamics.

doi: 10.1016/j.intimp.2023.109917

Figure Lengend Snippet: Fig. 1. USP25 was associated with T cell activation in graft rejection (A and B) Flow cytometric analysis of the frequency of CD8 + or CD4 + T cells in the spleens (n = 3) and grafts (n = 3) of WT mice at 1 day, 4 days and 7 days after heart transplantation. (C and D) Representative images of hematoxylin and eosin (H&E) and Immunofluorescence stained sections of heart allografts harvested from each groups at 1 day, 4 days and 7 days after heart transplantation. Scale bar = 100 μm and 20 μm.(E) qPCR analysis of mRNA expression of indicated genes in T cells of spleens at 1 day and 7 days after heart transplantation (n = 3). (F) Immunoblot analysis of the indicated proteins in T cells stimulated with anti-CD3 and anti-CD28. The data are representative of three or more independent ex periments and are presented as the mean ± SD. The P value in (A、B and E) was analyzed by two-tailed unpaired Student’s t test. ns, not statistically significant; *, P < 0.05.

Article Snippet: For cell transfusion, each Usp25-/- mouse was injected with 4 × 106 CD3+ T lymphocytes or CD3lymphocytes in 100 μl PBS which were sorted from C57BL/6 mice using Miltenyi Biotec CD3ε MicroBead Kit by tail vein injection before heart transplantation.

Techniques: Activation Assay, Transplantation Assay, Immunofluorescence, Staining, Expressing, Western Blot, Two Tailed Test

Journal: International immunopharmacology

Article Title: USP25 deficiency promotes T cell dysfunction and transplant acceptance via mitochondrial dynamics.

doi: 10.1016/j.intimp.2023.109917

Figure Lengend Snippet: Fig. 3. USP 25-deficiency mice showed delayed skin allograft rejection (A) Skin allograft appearance of WT (upper panel) and Usp25–/– (lower panel) mice from day 6 to 23 post-transplantation. (B) The graft survival rate in WT and Usp25–/– mice within 25 days post-transplantation (n = 3). (C) Flow cytometric analysis of WT and Usp25–/– CD4 + and CD8 + T cells in the spleens of WT mice at 7 days after heart transplantation (n = 6). (D and E) Representative images of hematoxylin and eosin (H&E) and Immunofluorescence stained sections of CD3 + T cells in allografts harvested from WT and Usp25–/– mice at 7 days after skin transplantation. Scale bar = 100 μm and 50 μm. The data are representative of three or more independent experiments and are presented as the mean ± SEM. The P value in (B) was analyzed by log-rank test; The P value in (C) was analyzed by two-tailed unpaired Student’s t test. ns, not statistically significant; *, P < 0.05.

Article Snippet: For cell transfusion, each Usp25-/- mouse was injected with 4 × 106 CD3+ T lymphocytes or CD3lymphocytes in 100 μl PBS which were sorted from C57BL/6 mice using Miltenyi Biotec CD3ε MicroBead Kit by tail vein injection before heart transplantation.

Techniques: Transplantation Assay, Immunofluorescence, Staining, Two Tailed Test

Journal: International immunopharmacology

Article Title: USP25 deficiency promotes T cell dysfunction and transplant acceptance via mitochondrial dynamics.

doi: 10.1016/j.intimp.2023.109917

Figure Lengend Snippet: Fig. 4. Usp25-/- T cell showed dysfunction and impaired cytotoxic ability (A) The graft survival rate in Usp25–/– mice with the transfusion of CD3+ T lym phocytes (4 × 106cells) or CD3- lymphocytes (4 × 106cells) before transplantation. (B) T cells from WT and Usp25–/– mice were cocultured with BMDCs from BALB/c with the ratio 5:1 for 18 h. Production of IFN-gamma by flow cytometric analysis gated on CD4 + and CD8 + T-cell populations in the mixed lymphocyte reaction (MLR); histogram shows the Production of IFN-gamma after different treatments (n = 3–4). (C) The serum levels of TNF-α, IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-13 and IL- 17 of WT and Usp25–/– mice were measured at 7 days after heart transplantation by CBA (n = 6). (D) The supernatant levels of TNF-α and IFN-γ from T cells stimulated with anti-CD3 and anti-CD28 of WT and Usp25–/– mice were measured by Elisa. (E) Flow cytometric analysis of Granzyme B + and Perforin + T cells in the spleens and grafts of WT and Usp25–/– mice at 7 days after heart transplantation (n = 3). (F) Representative CFSE profile of CD3 + T cells stimulated with anti-CD3 and anti-CD28. (G) Flow cytometric analysis of Granzyme B + and Perforin + T cells stimulated with anti-CD3 and anti-CD28 of WT and Usp25–/– mice (n = 4). The data are representative of three or more independent experiments and are presented as the mean ± SD. The P value in (A) was analyzed by log-rank test; The P value in (B, C, D, E and G) was analyzed by two-tailed unpaired Student’s t test. ns, not statistically significant; *, P < 0.05.

Article Snippet: For cell transfusion, each Usp25-/- mouse was injected with 4 × 106 CD3+ T lymphocytes or CD3lymphocytes in 100 μl PBS which were sorted from C57BL/6 mice using Miltenyi Biotec CD3ε MicroBead Kit by tail vein injection before heart transplantation.

Techniques: Transplantation Assay, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: International immunopharmacology

Article Title: USP25 deficiency promotes T cell dysfunction and transplant acceptance via mitochondrial dynamics.

doi: 10.1016/j.intimp.2023.109917

Figure Lengend Snippet: Fig. 5. USP25 interacts with ATP5a1 and affects mitochondrial related ATP synthesis (A) Interaction between USP25 and ATP5a1 in T cells determined by co- immunoprecipitation analysis using rabbit IgG or anti-USP25 antibodies. (B) Immunoblot analysis of the indicated proteins in T cells stimulated with anti-CD3 and anti-CD28 and T cells from the spleens after heart transplantation. (C) TEM sections of mitochondria in WT and KO T cells of grafts at 7 days after heart trans plantation. Scale bars = 1 μm and 500 nm. (D) Flow cytometric analysis of mitochondria labeled with Mito Tracker Green in WT and Usp25–/– T cells from the grafts after heart transplantation and stimulated with anti-CD3 and anti-CD28 (n = 3). (E) Fluorescence intensity of CD3 + T cells from the grafts and spleens of WT and Usp25–/– mice at 7 days after heart transplantation after loading with DCFH-DA (n = 3). (F) Cellular ATP levels were decreased in T cells from the grafts in Usp25-/-

Article Snippet: For cell transfusion, each Usp25-/- mouse was injected with 4 × 106 CD3+ T lymphocytes or CD3lymphocytes in 100 μl PBS which were sorted from C57BL/6 mice using Miltenyi Biotec CD3ε MicroBead Kit by tail vein injection before heart transplantation.

Techniques: Immunoprecipitation, Western Blot, Transplantation Assay, Labeling, Fluorescence

Journal: International immunopharmacology

Article Title: USP25 deficiency promotes T cell dysfunction and transplant acceptance via mitochondrial dynamics.

doi: 10.1016/j.intimp.2023.109917

Figure Lengend Snippet: Fig. 6. ATP supplementation changed the mass and morphology of Usp25–/– mitochondria (A) Flow cytometric analysis of mitochondria labeled Mito Tracker Green in T cells stimulated with anti-CD3 and anti-CD28 of WT, Usp25–/–, WT with ATP supplement and Usp25–/– with ATP supplement in vitro. (B) Flow cytometric analysis of Granzyme B + and Perforin + T cells and mitochondria labeled with Mito Tracker Green in WT and Usp25–/– T cells stimulated with anti-CD3 and anti- CD28 and different concentrations of ATP. (C) TEM sections of mitochondria in WT T cells and KO T cells with daily ATP injection of grafts at 7 days after heart transplantation. Scale bars = 500 nm. (D) ATP5a1 ubiquitination assays in 3 T3 cells transfected with ATP5a1, USP25 and Ub-K48.

Article Snippet: For cell transfusion, each Usp25-/- mouse was injected with 4 × 106 CD3+ T lymphocytes or CD3lymphocytes in 100 μl PBS which were sorted from C57BL/6 mice using Miltenyi Biotec CD3ε MicroBead Kit by tail vein injection before heart transplantation.

Techniques: Labeling, In Vitro, Injection, Transplantation Assay, Ubiquitin Proteomics, Transfection