Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: ( A – D ) Irradiated NSG mice were transplanted with MPB CD34 + HSPC that were transduced with a GFP-encoding LV vector (gene transfer 63%–66%) either i.v. or i.b.m. at the indicated cell doses. Human cell engraftment was analyzed by determining the percentage of human CD45 + (hCD45 + ) cells and GM hCD45 + (hCD45 + GFP + ) cells in the BM obtained via BM aspirate at 12 weeks to assess short-term engraftment ( A and B ) and from pooled BM from femurs, tibias, and iliac crests at 24 weeks to determine long-term human engraftment ( C and D ). Bars represent median engraftment ± 95% CI. Each symbol represents an individual mouse; n = 18–20 mice per cell dose per transplant method. Mice were transplanted using 4 unique MPB donors in 4 experiments; statistical analysis was performed using 1-way ANOVA comparing i.v. and i.b.m. groups at each cell dose with correction for multiple comparisons. ( E – H ) Lineage output of the human xenograft. BM was stained with antibodies specific for T cell lineage (hCD3), B cell lineage (hCD19), myeloid lineage (hCD33), and HSPC (hCD34) to determine the lineage output of the transplanted total HSPC ( E and G ) and GM HSPC ( F and H ) at 12 weeks and 24 weeks after transplant. Statistical analysis was performed using 2-way ANOVA. * P < 0.05, ** P < 0.01.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Irradiation, Transduction, Plasmid Preparation, Staining

Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: ( A ) MPB CD34 + HSPC were injected i.v., i.b.m. using a standard syringe, or slow i.b.m. using a Hamilton syringe over 1 minute, and homing of CD34 + cells to the BM was analyzed 20–22 hours later. To determine if mechanical pressure in the injected femur alters homing, irradiated CD34 – cells were injected i.b.m., and CD34 + cells were injected i.v. into the same animal. Homing was determined in the injected femurs (IF) and non-IF with i.b.m. injections, and in both femurs (BF) combined with i.v. injection. Symbols represent individual animals; n = 3 mice per group; statistical analysis was performed using 2-way ANOVA. ( B and C ) For i.b.m. injections, BM from the IF and non-IF was analyzed for expression of CXCL12 and VCAM1 on mouse stromal cells (hCD45 – PKH-26 – , mouse lineage – mCD45 – mCD51 + cells). n = 5 mice per group; data were normalized to i.v. injected group; statistical analysis was performed by Mann-Whitney U test. ( D and E ) CXCR4 and VLA4 homing receptor expression of CD34 + HSPC, CD34 + CD38 + HPC, and CD34 + CD38 – CD90 + HSC-enriched populations. Data were normalized to CXCR4 and VLA4 expression on CD34 + HSPC. Symbols represent unique MPB donors; n = 5 per group; statistical analysis was performed using ANOVA. ( F ) Adhesion of CD34 + HSPC, CD34 + CD38 + HPC, and CD34 + CD38 – CD90 + HSC to CXCL12. Symbols represent unique MPB donors; n = 5 per group; statistical analysis was performed using ANOVA. ( G ) Binding of CD34 + HSPC, CD34 + CD38 + HPC, and CD34 + CD38 – CD90 + HSC to the VLA4 affinity detection ligand LDV-FITC. Data were normalized to LDV-FITC expression in CD34 + HSPC. Symbols represent unique MPB donors; n = 3 per group; statistical analysis was performed using 1-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001. In B – E and G , data were normalized to the indicated group, to account for variability in MFI between different experiments/donors.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Injection, Irradiation, Expressing, MANN-WHITNEY, Binding Assay

Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: ( A – C ) High CXCR4 expression on HPC mediates their preferential homing locally with i.b.m. transplant; CD34 + HSPC were transduced with a BFP LV vector, and the CD34 + CD38 + BFP + HPC were sorted by flow cytometry 72 hours after gene transfer. HPC were blocked with AMD3100 (CXCR4 antagonist) or BIO5192 (VLA4 antagonist) before i.v. or i.b.m. delivery ( A ). Homing of hCD34 + cells was analyzed in the injected femur (IF) and non-IF for i.b.m. injected mice and in both femurs combined in i.v. injected mice. Data were normalized to i.v. injected control CD34 + CD38 + cells, and the fold increase is indicated within the bar ( B and C ). n = 5–6 mice per group; statistical analysis was performed by 1-way ANOVA. ( D – F ) Induction of high CXCR4 expression on CD34 + CD38 – HSC enriched population via gene transfer confers them with a homing advantage despite higher abundance of CD34 + CD38 + HPC. CD34 + HSPC were transduced with a BFP LV vector or a BFP-CXCR4 LV vector and sorted for transduced CD34 + CD38 – BFP + cells and untransduced CD34 + CD38 + BFP – HPC at 72 hours. Transduced CD34 + CD38 – cells were mixed with untransduced CD34 + CD38 + cells and transplanted i.v. or i.b.m., and homing of CD34 + CD38 – BFP + cells into BM was analyzed at 20–22 hours ( D ). CXCR4 expression on injected cells at time of transplant is shown and the MFI on injected cells at time of transplant was as follows: CD34 + CD38 – control, 16,930; CD34 + CD38 + control, 35,098; and CD34 + CD38 – CXCR4 transduced, 37,453. ( E ). Homing in BM is shown ( F ). n = 3–10 mice per experimental arm; statistical analysis was performed using 1-way ANOVA. * P < 0.05.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Expressing, Transduction, Plasmid Preparation, Flow Cytometry, Injection, Control

Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: CD34 + CD38 – HSC-enriched cells were obtained using immunomagnetic sorting, transduced with a GFP LV vector (gene transfer efficiency was 69%), and transplanted into NSG mice i.v. or i.b.m. in 2 limiting dilution doses. ( A and B ) Long-term (24 week) engraftment of total (hCD45 + ) and GM human (hCD45 + GFP + ) cells is shown. Symbols represent individual animals; n = 3–4 mice per treatment condition; statistical analysis was performed by comparing the modes of delivery comparing the different cell doses using 1-way ANOVA. * P < 0.05. ( C and D ) Lineage output at 24 weeks shows that the long-term human graft was multilineage, composed of B, T, and myeloid cells and CD34 + HSPC. Statistical analysis was performed using 2-way ANOVA.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Transduction, Plasmid Preparation

Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: ( A ) Construct design. The aa sequence of VPR and its mutant version generated is shown. VPR, a small HIV-1 accessory protein, is carried in viral particles bound to gag via residues in its central region that fold into 3 α-helices. The C-terminal domain has 6 arginine residues that potentiate nuclear localization, G 2 M arrest, and apoptosis. S79 phosphorylation is important for cell cycle arrest. We truncated VPR at the 78 aa and mutated VPR R77Q . W54 and Q65 interacts with DNA damage response (DDR) proteins via UNG2 and DCAF and were mutated to VPR W54R and VPR Q65R . The triple mutated and truncated VPR (VPR MT ) would lack pathogenicity (residues known to be associated with VPR toxicity are highlighted in red) but allow binding to gag. VPR MT was fused to CXCR4 cDNA via the HIV-1 protease cleavage site (PCS) to generate VPR MT -CXCR4. ( B ) K562 cells were transduced with LV CXCR4 vector-like particles (VLP; empty vector particles lacking the vector genome) at increasing particle concentration and analyzed for CXCR4 expression using flow cytometry. Mean fluorescence intensity (MFI) of CXCR4 is listed against volume of VLP added. ( C and D ) A GFP-encoding LV was either packaged using standard packaging plasmids (LV, black) or packaged with VPR MT -CXCR4 plasmid in addition in order to package the CXCR protein attached to the LV capsid (LV CXCR4 , red). CXCR4 expression on MPB CD34 + cells transduced with LV CXCR4 vector compared with cells transduced with the control LV vector 24 hours following gene transfer is shown. n = 4; statistical analysis was performed by Mann-Whitney U test. ( E ) The time course of CXCR4 expression in LV CXCR4 HSPC, normalized to that of control LV HSPC, is shown, with expression peaking at 24 hours that returns to baseline by 72 hours. Symbols represent individual MPB donors; n = 3.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Construct, Sequencing, Mutagenesis, Generated, Phospho-proteomics, Binding Assay, Transduction, Plasmid Preparation, Concentration Assay, Expressing, Flow Cytometry, Fluorescence, Control, MANN-WHITNEY

Journal: JCI Insight

Article Title: Differential CXCR4 expression on hematopoietic progenitor cells versus stem cells directs homing and engraftment

doi: 10.1172/jci.insight.151847

Figure Lengend Snippet: Experimental schema for assessment of homing and engraftment of CD34 + CD38 – cells transduced with GFP LV or GFP LV CXCR4 that were transplanted into NSG mice either via i.v. or i.b.m. delivery. ( A ) The number of animals used for the homing experiment and engraftment experiments is indicated under each experimental arm. ( B ) Homing of CD34 + CD38 – cells in the BM. Data were normalized to i.v. injected control CD34 + CD38 – cells, and the fold increase is indicated within the bar. Symbols represent individual mice; statistical analysis was performed using ANOVA. ( C and D ) Long-term engraftment of CD34 + CD38 – cells was assessed by determining the percentage of human CD45 + GFP – cells ( C ) and human CD45 + GFP + ( D ) cells 24 weeks following transplant. Data were normalized to i.v. transplanted GFP LV transduced CD34 + CD38 – cells, and the fold increase is indicated within the bar. Gene transfer was 60% with GFP LV and 52% with GFP LV CXCR4 . Symbols represent individual mice; statistical analysis was performed by 1-way ANOVA. * P < 0.05, ** P < 0.01.

Article Snippet: CD34 + CD38 – cells were isolated from G-CSF MPB by magnetic selection with human CD34 + CD38 – Isolation Kit (Miltenyi Biotec, 130-114-822) and cryopreserved.

Techniques: Transduction, Injection, Control

Journal: Cancers

Article Title: Neoantigen Dendritic Cell Vaccination Combined with Anti-CD38 and CpG Elicits Anti-Tumor Immunity against the Immune Checkpoint Therapy-Resistant Murine Lung Cancer Cell Line LLC1

doi: 10.3390/cancers13215508

Figure Lengend Snippet: The adenosine pathway in the LLC1 tumor microenvironment. ( A ) RNA was extracted from ASB-XIV and LLC1 tumor cells and subjected to RNA-Seq analysis. The mRNA expressions of immunosuppressive molecules in these two cell lines were shown. ( B ) The expressions of CD38, CD39, and CD73 on LLC1 were examined by flow cytometry in vitro. ( C ) C57BL/6 mice ( n = 5) received a subcutaneous injection of 5 × 10 5 LLC1. The combination of 2 mg/Kg A2A and 1 mg/Kg A2B adenosine receptor antagonists (SCH 58261 and PSB 1115) was administered on days 7, 9, 11, 13, 15, 17, and 19. ( D ) LLC1 tumor growth was compared in mice ( n = 8) that received 30μg CpG monotherapy, L82 LP-pulsed DC vaccination (1 × 10 6 ), and CpG, or the triple combination of DC vaccination, CpG, and A2R antagonists. DC vaccines were prophylactically administered twice at a 2-week interval.

Article Snippet: Monoclonal antibodies (mAbs) specific for PD-1 (RMP1-14), CTLA-4 (9H10), CD38 (NIMR5), and CD16/32 (2.4G2) were purchased from BioXcell (Lebanon, NH, USA).

Techniques: RNA Sequencing Assay, Flow Cytometry, In Vitro, Injection

Journal: Cancers

Article Title: Neoantigen Dendritic Cell Vaccination Combined with Anti-CD38 and CpG Elicits Anti-Tumor Immunity against the Immune Checkpoint Therapy-Resistant Murine Lung Cancer Cell Line LLC1

doi: 10.3390/cancers13215508

Figure Lengend Snippet: The triple combination of DC vaccination, CpG and anti-CD38 mAb treatment inhibited LLC1 tumor growth. ( A ) C57BL/6 mice were divided into five groups (five mice per group): (1) 30μg CpG treatment; (2) CpG plus 250μg anti-CD38 mAb; (3) 1×10 6 L82 LP-pulsed DC vaccination with CpG; (4) the triple combination of L82 LP-pulsed DC vaccination, CpG, and anti-CD38 mAb; and (5) the quadruple combination of DC, CpG, anti-CD38 mAb, and 200μg of anti-PD-1 mAb. LLC1 cells (5 × 10 5 ) were subcutaneously inoculated and tumor growth was monitored. ( B ) On day 14, tumor-infiltrating cells were extracted from these mice ( n = 5) and subjected to flow cytometry. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: Monoclonal antibodies (mAbs) specific for PD-1 (RMP1-14), CTLA-4 (9H10), CD38 (NIMR5), and CD16/32 (2.4G2) were purchased from BioXcell (Lebanon, NH, USA).

Techniques: Flow Cytometry

Journal: bioRxiv

Article Title: Non-invasive imaging of cell-based therapies using acoustic reporter genes

doi: 10.1101/2024.11.01.621111

Figure Lengend Snippet: a , Schematic of lentiviral vectors designed to report T cell activity state by expressing GVs downstream of the NFAT promoter. T cell activation is chemically induced by incubating cells with PMA and ionomycin, which trigger the NFAT promoter to express rtTA, and in the presence of doxycycline, activate transcription of GV genes. b , Representative BURST images (top) and signal quantification (bottom) of Jurkat cells transduced with the vectors in panel ( a ) with or without chemical activation, normalized to wild-type (WT) cells (not shown). c , Percentage of Jurkat cells in panel ( b ) expressing both fluorescent reporters (GFP and BFP), with and without activation. d , Imaging T cell activation in response to CD19-CD3 BTE-mediated engagement with CD19+ Raji cells, using cells engineered with the vectors shown in ( a ). e , Representative BURST images (top) and signal quantification (bottom) of Jurkat cells engineered to express GVs upon receptor-mediated activation in the presence of Dox. f , Percentage of cells from panel ( e ) expressing both fluorescent reporters (GFP and BFP), with and without receptor engagement. Statistical comparisons were performed using Fisher’s LSD test, with each condition compared to resting state T cells (without PMA/ionomycin or BTE stimulation). P-value for ( e ): 0.0173. All other p-values < 0.0001. Significance levels: *p<0 . 05, **p<0 . 01, ***p<0 . 001, ****p<0 . 0001 . Error bars represent mean ± s.e.m. of N = 3 biological replicates. Each data point represents the arithmetic mean of N = 2 technical replicates. Scale bars for all ultrasound images represent 1 mm.

Article Snippet: To induce receptor-mediated T cell cytotoxicity, CD19-CD3 BTE (1 ng/mL, BPS Bioscience) and IL-2 (100 U/mL) were added.

Techniques: Activity Assay, Expressing, Activation Assay, Transduction, Imaging

Journal: bioRxiv

Article Title: Non-invasive imaging of cell-based therapies using acoustic reporter genes

doi: 10.1101/2024.11.01.621111

Figure Lengend Snippet: a , Workflow for engineering T cells isolated from human PBMCs to express doxycycline-inducible GVs and imaging BTE-mediated homing of cytotoxic T cells into target tumors. Top: T cells are transduced with the 3-vector lentiviral system, and GFP expressing cells are sorted to select for transduction of the assembly factor vectors. The cells are then expanded in vitro for downstream characterization and in vivo injections. Bottom: T cells are systemically administered to immunocompromised mice bearing subcutaneous CD19+ Raji cell tumors, accompanied by intraperitoneal (IP) injections of BTE every 48 hours and IP injections of doxycycline for 72 hours prior to ultrasound imaging. b , BURST SBR (left) and representative images (right) of GV-expressing and WT T cells. Paired one-tailed t test, p = 0.0115. Error bars represent mean ± s.e.m. of N = 5 PBMC donors; each data point is the arithmetic mean of N = 3 technical replicates. c , Percentage of live Raji cells in a cytotoxicity assay, where T cells are co-cultured with CD19+ Raji cells at effector-to-target (E:T) ratios of 1:1 and 4:1. Welch’s two-tailed t-test, N = 6 biological replicates (2 PBMC donors and 3 replicates per donor). d , GFP and BFP fluorescence measurements from ( c ), normalized to the maximum mean fluorescence intensity (MFI) of each reporter. e , BURST images overlaid on anatomical B-mode grayscale images of representative Raji tumors in mice injected with GV-expressing T cells, either Dox-induced or uninduced, and mice injected with WT T cells. Red lines indicate tumor boundary used for signal quantification. f , Quantification of BURST signal inside tumors. Welch’s two-tailed t test, Dox+ vs. Dox-, p = 0.0067; Dox+ vs. WT, p = 0.0063. N = 10 mice for Dox+, N = 6 for Dox-, N = 6 for WT. g , Histology of an example tumor infiltrated by GV-expressing T cells and its corresponding BURST image. White: Raji-Antares cells; Magenta: CD8+ cytotoxic T cells; Green: GFP+ T cells; Blue: BFP+ T cells. h , Schematic of T cell distribution in the Dox-induced tumor from ( g ). i , Correlation of the BURST signal within tumors with the absolute numbers of GFP+ and BFP+ T cells in corresponding histological sections. Pearson correlation: r = 0.89, p = 0.0072, N = 7 mice. One mouse (gray data point) was excluded due to a significant number of T cells located outside the imaging plane whose GV expression was not captured by BURST (see ). All scale bars represent 1 mm.

Article Snippet: To induce receptor-mediated T cell cytotoxicity, CD19-CD3 BTE (1 ng/mL, BPS Bioscience) and IL-2 (100 U/mL) were added.

Techniques: Isolation, Imaging, Transduction, Plasmid Preparation, Expressing, In Vitro, In Vivo, One-tailed Test, Cytotoxicity Assay, Cell Culture, Two Tailed Test, Fluorescence, Injection