Journal: Cell Reports Medicine

Article Title: Identification of cells of leukemic stem cell origin with non-canonical regenerative properties

doi: 10.1016/j.xcrm.2024.101485

Figure Lengend Snippet: RECs demonstrate no stemness capacity and co-localize to CD34 + cells within leukemic tissue (A) Representative flow plot of hCD45 and CD33 expression in BM aspirates from PDXs 8 weeks post intra-femoral injection with RECs (n = 11, N = 3). (B) Bar graph of mean ± SD of CFU frequency (#colonies/cells seeded) of FACS-purified CD74 + /CD68 + cells and bulk AML patient MNCs normalized to average AML patient MNCs. (C) Bar graph of leukemic mutation VAF of FACS-purified CD74 + /CD68 + cells compared with control MNCs. (D) Whole H&E-stained tissue and representative images with and without spot overlays of BM tissue from AML patient 11 and BM donor 4. Scale bar, 100 μM. (E) Spots of CD74 + /CD68 + /CD34 + co-expression overlayed onto whole tissue section of AML BM11, and four representative images each from areas with and without CD74 + /CD68 + /CD34 + co-expression. Scale bars, 50 μM. (F) Xenograft BM sections of engrafted CB and AML, with hCD74 hCD68 hCD34 immunofluorescent labels by MIBI-TOF methodology. Examples of CD74 + /CD68 + cells and CD34 + are highlighted in white. (G) Bar graph of the mean +/- SD distance between CD74+/CD68+ and CD34 + cells in the AML vs. CB xenograft BM (∗∗∗∗p < 0.0001, Student’s t test). See also Figure S5 , Table S1 .

Article Snippet: PE mouse anti-human CD33 , BD Biosciences , Cat#347787; RRID: AB_400350.

Techniques: Expressing, Injection, Purification, Mutagenesis, Staining

Journal: Cell Reports Medicine

Article Title: Identification of cells of leukemic stem cell origin with non-canonical regenerative properties

doi: 10.1016/j.xcrm.2024.101485

Figure Lengend Snippet:

Article Snippet: PE mouse anti-human CD33 , BD Biosciences , Cat#347787; RRID: AB_400350.

Techniques: Recombinant, Selection, Software

Journal: bioRxiv

Article Title: Expansion of human hematopoietic stem cells by inhibiting translation

doi: 10.1101/2023.11.28.568925

Figure Lengend Snippet: A. Limiting dilution analysis (LDA) of human UCB CD34 + cells at day 0 or after 7 days of culture in CRCY+DMSO or CRCY with 4E1RCat (2 µM). Dose of cells injected is based on the number of CD34 + cells on day 0. Chimerism was measured as human CD45 + cells at 20 weeks in bone marrow with engraftment defined as ≥ 0.1% hCD45 + . Data are from 2 distinct LDA/transplant experiments. B. HSC frequency with 95% CI calculated with ELDA software. p = 0.001 for CRCY+4E1RCat compared to fresh cells. No significant difference for CRCY+DMSO compared to Day 0 cells. C-F. The percentage of human T cells (CD3 + cells), B cells (CD19 + cells), myeloid cells (CD33 + cells), and lineage negative (CD34 + CD38 - ) cells is shown for recipients injected with cells from ex vivo cultures corresponding to 5000, 500, 100, 25 CD34 + cells on day 0. G. Scheme for secondary transplantation from primary recipients that had received day 0 cells or cells cultured in CRCY+4E1RCat for 7 days. H. The percentage engraftment of human CD45 + cells in peripheral blood (16 weeks) in secondary recipients. I. The percentage engraftment of human CD45 + cells in the bone marrow (18 weeks) in secondary recipients. J-N. The percentage (log 10 scale) of human lineage negative cells (CD34 + CD38 - ), B cells (CD19 + ), myeloid cells (CD33 + ), megakaryocytes (CD41 + ) and erythroid cells (GlyA + ) in the bone marrow (18 weeks) of secondary recipients. Samples with 0% engraftment are shown at the baseline (with broken y-axes),

Article Snippet: After 18 weeks, bone marrow was collected and analyzed with PE anti-human CD45 antibody (CAT# 368510, Biolegend), APC/Cyanine7 anti-mouse CD45 antibody (CAT# 103116, Biolegend), APC anti-human CD3 antibody (CAT# 317318, Biolegend), BB515 Mouse anti-Human CD33 (CAT# 564588, BD Biosciences), BB700 mouse anti-Human CD19 (CAT# 566396, BD Biosciences), Brilliant Violet 421™ anti-human CD41 antibody (CAT# 303730, Biolegend), BUV395 Mouse anti-Human CD235a antibody (CAT# 563810, BD Biosciences), PE/Cyanine7 anti-human CD34 antibody (CAT# 343616, Biolegend), and Brilliant Violet 711™ anti-human CD38 antibody (CAT# 303528, Biolegend).

Techniques: Injection, Software, Ex Vivo, Transplantation Assay, Cell Culture

Journal: bioRxiv

Article Title: Expansion of human hematopoietic stem cells by inhibiting translation

doi: 10.1101/2023.11.28.568925

Figure Lengend Snippet: The percentage of human CD45 + cells, T cells (CD3 + ), B cells (CD19 + ), myeloid cells (CD33 + ), and CD34 + CD38 - cells in each primary recipient mouse at 20 weeks in bone marrow are represented by a heatmap. Red indicates percentage engraftment (human cells) is > 0.1%. Blue indicates human cell engraftment < 0.1%. White indicates percentage engraftment = 0.1%. Data show results from expansion/transplants using 2 distinct mixed donor UCB samples.

Article Snippet: After 18 weeks, bone marrow was collected and analyzed with PE anti-human CD45 antibody (CAT# 368510, Biolegend), APC/Cyanine7 anti-mouse CD45 antibody (CAT# 103116, Biolegend), APC anti-human CD3 antibody (CAT# 317318, Biolegend), BB515 Mouse anti-Human CD33 (CAT# 564588, BD Biosciences), BB700 mouse anti-Human CD19 (CAT# 566396, BD Biosciences), Brilliant Violet 421™ anti-human CD41 antibody (CAT# 303730, Biolegend), BUV395 Mouse anti-Human CD235a antibody (CAT# 563810, BD Biosciences), PE/Cyanine7 anti-human CD34 antibody (CAT# 343616, Biolegend), and Brilliant Violet 711™ anti-human CD38 antibody (CAT# 303528, Biolegend).

Techniques:

Journal: bioRxiv

Article Title: Expansion of human hematopoietic stem cells by inhibiting translation

doi: 10.1101/2023.11.28.568925

Figure Lengend Snippet: A-E. The percentage of human CD45 + cells, CD34 + CD38 - cells, myeloid cells (CD33 + ), T cells (CD3 + ), and B cells (CD19 + ) at day 29 post-transplant in bone marrow of mice used as donors for secondary transplant. Mice had received either uncultured (Day 0) CD34 + cells or cells cultured in CRCY+4E1RCat for 7 days. F. Table showing percent engraftment of each population in each mouse at 29 weeks.

Article Snippet: After 18 weeks, bone marrow was collected and analyzed with PE anti-human CD45 antibody (CAT# 368510, Biolegend), APC/Cyanine7 anti-mouse CD45 antibody (CAT# 103116, Biolegend), APC anti-human CD3 antibody (CAT# 317318, Biolegend), BB515 Mouse anti-Human CD33 (CAT# 564588, BD Biosciences), BB700 mouse anti-Human CD19 (CAT# 566396, BD Biosciences), Brilliant Violet 421™ anti-human CD41 antibody (CAT# 303730, Biolegend), BUV395 Mouse anti-Human CD235a antibody (CAT# 563810, BD Biosciences), PE/Cyanine7 anti-human CD34 antibody (CAT# 343616, Biolegend), and Brilliant Violet 711™ anti-human CD38 antibody (CAT# 303528, Biolegend).

Techniques: Cell Culture

Journal: bioRxiv

Article Title: Expansion of human hematopoietic stem cells by inhibiting translation

doi: 10.1101/2023.11.28.568925

Figure Lengend Snippet: Bone marrow harvested from primary recipients at 29 weeks was transplanted into conditioned secondary recipients as described in . The percentage of human CD45 + cells was measured in peripheral blood (PB) at 16 weeks. Bone marrow (BM) was harvested at 18 weeks and the percentage of human CD45 + cells T cells (CD3 + ), B cells (CD19 + ), myeloid cells (CD33 + ), megakaryocytic cells (CD41 + ), erythroid cells (GlyA + ) and CD34 + CD38 - cells was measured.

Article Snippet: After 18 weeks, bone marrow was collected and analyzed with PE anti-human CD45 antibody (CAT# 368510, Biolegend), APC/Cyanine7 anti-mouse CD45 antibody (CAT# 103116, Biolegend), APC anti-human CD3 antibody (CAT# 317318, Biolegend), BB515 Mouse anti-Human CD33 (CAT# 564588, BD Biosciences), BB700 mouse anti-Human CD19 (CAT# 566396, BD Biosciences), Brilliant Violet 421™ anti-human CD41 antibody (CAT# 303730, Biolegend), BUV395 Mouse anti-Human CD235a antibody (CAT# 563810, BD Biosciences), PE/Cyanine7 anti-human CD34 antibody (CAT# 343616, Biolegend), and Brilliant Violet 711™ anti-human CD38 antibody (CAT# 303528, Biolegend).

Techniques:

Journal: Cell Reports Medicine

Article Title: Oncolytic virus M1 functions as a bifunctional checkpoint inhibitor to enhance the antitumor activity of DC vaccine

doi: 10.1016/j.xcrm.2023.101229

Figure Lengend Snippet: Tumor cells inhibit DCs through SIRPα-CD47 (A) Protein kinase/phosphatase-related GSEA pathways significantly enriched in B16-F10-cocultured DCs (co-DCs) compared with untreated DCs (iDCs). (B) qPCR analysis comparing iDCs and co-DCs cocultured with B16-F10 cells for 48 h. These genes participate in the ITIM/ITAM downstream pathway; n = 3. (C) Representative western blot pictures (left) and quantitative statistics (right) of the phosphorylation of SIRPα-related downstream kinases; n = 3. (D) Heatmap of top 10 changes in ITIM-containing receptors between the co-DC and the iDC group. (E) The immune checkpoint expression changes between co-DCs and the untreated iDC group; n = 6 in SIRPα, PD-1, and CTLA-4; n = 5 in TIM-3; n = 3 in PIR-B and CD33. (F) SIRPα-KO mouse- or wild-type (WT) mouse-derived DCs were cocultured with B16-F10 cells, and the maturation of CD11c + DCs was determined by flow cytometry; n = 3. (G and H) WT mouse-derived DCs were cocultured with B16-F10-WT or B16-F10-CD47 KO cells at a 3:1 ratio for 48 h. The proportion of cells with high expression levels of CD80, CD86, and CD83 (G) and secretion levels of TNF-α (H) was determined to assess the DC activation/maturation phenotype; n = 3 in per group. (I–L) C57BL/6J mice were implanted subcutaneously in the right flank with B16-F10 cells on day 0 and administered one dose of vehicle, oncolysate-stimulated DC vaccine, or oncolysate-stimulated SIRPα-KO DC vaccine by intratumoral injection on day 6 (tumor volumes were approximately 50 mm 3 ). (I) The levels of TNF-α and IL-12 in the tumor interstitial fluid. On the fifth day after treatment, fresh tumor tissues were collected, weighed, and incubated at 37°C for 2 h in 1 mL of PBS per gram of tumor tissue to obtain the tumor interstitial fluid for ELISA; n = 5. (K) Tumor growth curves and (L) Kaplan-Meier survival curves are shown; n = 6. (M–O) C57BL/6J mice were implanted subcutaneously in the right flank with B16-F10-WT or B16-F10-CD47 KO cells on day 0 and administered one dose of vehicle or B16-F10 oncolysate-stimulated DC vaccine by intratumoral injection on day 6. (N) Tumor growth curves (n = 6) and (O) Kaplan-Meier survival curves (n = 7) are shown. The p values were determined by unpaired Student’s t test (B, C, E), one-way ANOVA (F–I), one-way ANOVA at the final time point (K, N), or log rank test (L, O). n.s., not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001.

Article Snippet: To detect changes in immune checkpoints on the surface of DCs after coculture with tumor cells, DCs were cocultured with B16-F10 tumor cells for 48 h and collected for staining with the following fluorescent antibodies: anti-Mouse CD11c PE Cyanine7, anti-Mouse SIRPα Alexa Fluor 700 (144022, BioLegend, USA), anti-Mouse CD33 PE (12-0331-82, eBioscience, USA), anti-Mouse PIR-A/B APC(17-3101-82, eBioscience, USA), anti-Mouse PD-1 APC (17-9981-82, eBioscience, USA), anti-Mouse TIM-3 PE (12-5870-82, eBioscience, USA), and anti-Mouse CTLA-4 PE (553720, BD, USA).

Techniques: Western Blot, Phospho-proteomics, Expressing, Derivative Assay, Flow Cytometry, Activation Assay, Injection, Incubation, Enzyme-linked Immunosorbent Assay

Journal: Cell Reports Medicine

Article Title: Oncolytic virus M1 functions as a bifunctional checkpoint inhibitor to enhance the antitumor activity of DC vaccine

doi: 10.1016/j.xcrm.2023.101229

Figure Lengend Snippet:

Article Snippet: To detect changes in immune checkpoints on the surface of DCs after coculture with tumor cells, DCs were cocultured with B16-F10 tumor cells for 48 h and collected for staining with the following fluorescent antibodies: anti-Mouse CD11c PE Cyanine7, anti-Mouse SIRPα Alexa Fluor 700 (144022, BioLegend, USA), anti-Mouse CD33 PE (12-0331-82, eBioscience, USA), anti-Mouse PIR-A/B APC(17-3101-82, eBioscience, USA), anti-Mouse PD-1 APC (17-9981-82, eBioscience, USA), anti-Mouse TIM-3 PE (12-5870-82, eBioscience, USA), and anti-Mouse CTLA-4 PE (553720, BD, USA).

Techniques: Control, Recombinant, Staining, Selection, Isolation, Red Blood Cell Lysis, Enzyme-linked Immunosorbent Assay, Reverse Transcription, SYBR Green Assay, RNA Sequencing, Software