Journal: Scientific Reports

Article Title: Identifying transcriptomic profiles of iron–quercetin complex treated peripheral blood mononuclear cells from healthy volunteers and diabetic patients

doi: 10.1038/s41598-024-60197-1

Figure Lengend Snippet: Box plots of the results of the flow cytometric analysis illustrating the proportions of cells expressing angiogenic and anti-inflammatory phenotypes, including CD3 + , CD4 + , CD8 + , CD11b + , CD14 + , CD31 + , CD34 + , CD45 + , CD105 + , CD192 + , CD206 + , and CD4 + /25 + . Statistical significance was assessed through ANOVA followed by Tukey's post-hoc test. In the plots, the presence of (*) denotes statistically significant differences, signifying a significance level of p < 0.05.

Article Snippet: Each 100 µL sample of the cell suspension was incubated with the appropriate IgG isotype controls for each fluorescence channel or with specific antibodies, including FITC-labeled anti-CD34 (Elabscience), FITC-labeled anti-CD14 (Miltenyi), FITC-labeled anti-CD11b (eBioscience), PE/Cy5.5‐labeled anti-CD45 (eBioscience), FITC-labeled anti-CD31 (Life Technologies), PE-labeled anti-CD105 (eBioscience), PE/Cy5.5‐labeled anti‐CD3(clone: UCHT1, Merck Millipore), APC‐labeled anti‐CD206 (clone 15–2, Sigma-Aldrich), APC‐labeled anti‐CCR2 (CD192, Sino Biological), PE‐labeled anti‐CD8a (clone: RPA‐T8, Merck Millipore), PE‐labeled anti‐CD4 (clone: RPA‐T4, Merck Millipore), and Alexa Fluor‐488‐labeled anti‐CD25 (Clone: BC96, eBioscience).

Techniques: Expressing

Journal: Frontiers in Immunology

Article Title: CEACAM 1, 3, 5 and 6 -positive classical monocytes correlate with interstitial lung disease in early systemic sclerosis

doi: 10.3389/fimmu.2022.1016914

Figure Lengend Snippet: Antibodies used for Mass Cytometry.

Article Snippet: CCR2 , K036C2 , 153Eu , Fluidigm.

Techniques: Cytometry

Journal: Nature Communications

Article Title: Lin − CCR2 + hematopoietic stem and progenitor cells overcome resistance to PD-1 blockade

doi: 10.1038/s41467-018-06182-5

Figure Lengend Snippet: CCR2 + HSCs increase T-cell activation within tumor after adoptive cell therapy. a Lineage negative HSCs were further isolated into stem and progenitor cell populations: Sca1 + HSCs, cKit + HSCs, CD133 + HSCs, CD38 + HSCs, CCR2 + HSCs. Each progenitor cell population was transferred with tumor-reactive T-cells (generated from GREAT mice) into tumor-bearing mice. Relative amounts of YFP + was measured in the tumors. b Late-stage tumor-bearing mice received adoptive T-cell therapy and dendritic cell (DC) vaccine with or without HSC transfer. Twenty-one days post transfer, brains were excised and sliced into sections, then analyzed for YFP + cells within the tumor microenvironment by measuring relative MFI across brain sections (* p = 0.0110, n = 5/group, Mann–Whitney test). c Late-stage tumor-bearing mice received adoptive T-cell therapy and DC vaccine with or without HSC transfer. Draining lymph nodes were harvested and YFP + CD3 + cells were enumerated to determine the relative amounts of activated T cells, * p = 0.0002, n = 5/group, Mann–Whitney test). d Two groups of KR158 glioma-bearing mice received adoptive T-cell therapy with concomitant transfer of either CCR2 − HSCs or CCR2 + HSCs. Tumors were excised and relative amounts of YFP + of CD3 + cells were measured with flow cytometry (* p = 0.0002, n = 5/group, unpaired t -test). e Tumor bearing mice were treated with either CCR2 + HSC + PD-1 or CCR2 − HSC + PD-1. Tumors were excised and RNA was isolated to conduct RT2 PCR Array for T- and B-cell activation (Qiagen). Genes represented demonstrated >2-fold change in expression (this has been replicated twice). All error bars represent s.e.m.

Article Snippet: CCR2 positive selection was conducted using biotinylated anti-mouse CCR2 antibody (Miltenyi Biotec cat. 130-108-721) followed by anti-biotin Microbead separation (Miltenyi Biotec clone Bio3-18E7.2).

Techniques: Activation Assay, Isolation, Generated, MANN-WHITNEY, Flow Cytometry, Expressing

Journal: Nature Communications

Article Title: Lin − CCR2 + hematopoietic stem and progenitor cells overcome resistance to PD-1 blockade

doi: 10.1038/s41467-018-06182-5

Figure Lengend Snippet: CCR2 + HSCs migrate to intracranial tumor and differentiate into dendritic cells. a Equal numbers of CCR2 − HSCs (isolated from GFP mice) and CCR2 + HSCs (isolated from DsRed mice) were injected into tumor-bearing mice. Relative amounts of cells derived from either GFP + CCR2 − HSCs and DsRed + CCR2 + HSCs were compared at 1 day (* p = 0.0010), 7 days (** p = 0.0013), and 30 days (*** p = 0.0003) post transfer (Mann–Whitney tests; n = 7 mice per group). b In a separate experiment, CCR2 + HSCs were isolated from GFP + mice and intravenously co-transferred into tumor-bearing mice that received adoptive cell therapy with tumor-reactive T-cells. Twenty-one days post transfer, GFP + cells found in the tumor were phenotyped for makers of dendritic cells. c , d Both CCR2 − HSCs and CCR2 + HSCs were isolated from GFP + mice and co-transferred into tumor-bearing mice that received adoptive cell therapy with tumor-reactive T-cells. Twenty-one days post transfer, GFP + cells found in the tumors were analyzed for markers of a suppressive phenotype. e CCR2 − HSCs and CCR2 + HSCs were isolated and cultured in vitro in dendritic cell media containing GM-CSF and IL-4. Resulting cells were electroporated with total tumor RNA and used as targets for tumor-reactive T-cells. Supernatant IFNγ was measured (* p = 0.0135, *n/s p = 0.5176, n = 5 replicates, unpaired t -test). All error bars represent s.e.m.

Article Snippet: CCR2 positive selection was conducted using biotinylated anti-mouse CCR2 antibody (Miltenyi Biotec cat. 130-108-721) followed by anti-biotin Microbead separation (Miltenyi Biotec clone Bio3-18E7.2).

Techniques: Isolation, Injection, Derivative Assay, MANN-WHITNEY, Cell Culture, In Vitro

Journal: Nature Communications

Article Title: Lin − CCR2 + hematopoietic stem and progenitor cells overcome resistance to PD-1 blockade

doi: 10.1038/s41467-018-06182-5

Figure Lengend Snippet: CCR2 + HSCs cross-prime endogenous T lymphocytes. a Either HSCs, CCR2 + HSCs, or CCR2 − HSCs isolated from DsRed + mice were stereotactically injected into tumors of glioma-bearing mice. DsRed+ cells were then observed in draining lymph nodes 1 week post transfer, and relative amounts of DsRed+ cells between groups was compared, CCR2 + HSCs vs. CCR2 − HSCs (* p = 0.021, Mann–Whitney, n = 5 mice per group). b Either HSCs, CCR2 + HSCs, or CCR2 − HSCs were isolated from DsRed + mice, then were stereotactically injected directly into tumors of glioma- or medulloblastoma-bearing mice followed by systemic administration of PD-1. Three weeks post transfer, draining lymph nodes were harvested and T-cells were isolated from the lymph nodes using magnetic bead isolation. These T-cells were then used as effector cells against tumor cell targets in a co-culture assay. This was conducted in KR158B glioma-bearing mice, and Ptc medulloblastoma-bearing mice, and IFNγ secretion was measured after the co-culture. T cells harvested from b KR158B glioma-bearing mice that received CCR2 + HSCs secreted an average of 1549.99 ± 212.621 pg/ml IFNγ, which is significantly more that T-cells derived from mice that received CCR2 − HSCs that contained 522.183 ± 87.32 pg/ml IFNγ (** p = 0.0111, two-tailed t -test). c T cells harvested from Ptc medulloblastoma-bearing mice that received CCR2 + HSCs secreted an average of 972.2 ± 110.6 pg/ml IFNγ, which is significantly more that T-cells derived from mice that received CCR2 − HSCs that contained 132.6 ± 20.74 pg/ml IFNγ (*** p = 0.0017, two-tailed t -test). All error bars represent s.e.m.

Article Snippet: CCR2 positive selection was conducted using biotinylated anti-mouse CCR2 antibody (Miltenyi Biotec cat. 130-108-721) followed by anti-biotin Microbead separation (Miltenyi Biotec clone Bio3-18E7.2).

Techniques: Isolation, Injection, MANN-WHITNEY, Co-culture Assay, Co-Culture Assay, Derivative Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Lin − CCR2 + hematopoietic stem and progenitor cells overcome resistance to PD-1 blockade

doi: 10.1038/s41467-018-06182-5

Figure Lengend Snippet: CCR2 + HSC-derived dendritic cells expand T-cells in adoptive cell therapy. a Experimental description. b Cells derived from GFP + HSCs, GFP + CCR2 − HSCs, or GFP + CCR2 + HSCs were isolated from the tumors of mice treated with adoptive cell therapy. These were then used as a vaccine in a second cohort of mice treated with adoptive cell therapy using DsRed + tumor-reactive T cells. Vaccine draining lymph nodes were harvested and relative amounts of DsRed + T-cells were measured between the group that received CCR2 + HSCs and the groups that received cells derived from CCR2 − HSCs (* p = 0.0001, t -test, n = 3 replicates per group). All error bars represent s.e.m.

Article Snippet: CCR2 positive selection was conducted using biotinylated anti-mouse CCR2 antibody (Miltenyi Biotec cat. 130-108-721) followed by anti-biotin Microbead separation (Miltenyi Biotec clone Bio3-18E7.2).

Techniques: Derivative Assay, Isolation

Journal: Nature Communications

Article Title: Lin − CCR2 + hematopoietic stem and progenitor cells overcome resistance to PD-1 blockade

doi: 10.1038/s41467-018-06182-5

Figure Lengend Snippet: CCR2 + HSCs increase efficacy of PD-1 against brain tumors. a KR158B tumor-bearing GREAT mice were treated with PD-1 and either CCR2 − HSCs or CCR2 + HSCs isolated from either wildtype C57BL/6 mice, MHC I −/− mice, or MHC II −/− mice. Tumors were excised and relative amounts of YFPP + CD3 + cells were measured, specifically between the group that received wildtype CCR2 + HSCs and CCR2 + HSCs from MHC I −/− (* p < 0.0001, Mann–Whitney, n = 5 mice per group). b KR258B tumor-bearing mice received no treatment, HSC only, PD-1 only, CCR2 − HSCs, CCR2 + HSCs, HSCs+PD-1, CCR2 − HSCs + PD-1, or CCR2 + HSCs + PD-1 (* p = 0.0233, ** p = 0.0006). c Ptc medulloblastoma tumor-bearing mice received no treatment, HSC only, PD-1 only, CCR2 − HSCs, CCR2 + HSCs, HSCs + PD-1, CCR2 − HSCs + PD-1, or CCR2 + HSCs + PD-1 (* p = 0.0001, ** p = 0.0005). d Mice that received lethal total body irradiation for myeloablative host conditioning received either no treatment, HSCs, CCR2 − HSCs, or CCR2 + HSCs, then followed for survival. e KR158B glioma-bearing mice received adoptive cell therapy (ACT) and either HSCs, CCR2 − HSCs, or CCR2 + HSCs, * p = 0.0005, *n/s p = 0.1776. f Ptc medulloblastoma-bearing mice received adoptive cellular therapy (ACT) and either HSCs or CCR2 + HSCs. All error bars represent s.e.m. Comparison of survival data was conducted with Gehan–Breslow–Wilcoxon test

Article Snippet: CCR2 positive selection was conducted using biotinylated anti-mouse CCR2 antibody (Miltenyi Biotec cat. 130-108-721) followed by anti-biotin Microbead separation (Miltenyi Biotec clone Bio3-18E7.2).

Techniques: Isolation, MANN-WHITNEY, Irradiation, Comparison

Journal: Theranostics

Article Title: Acellular cardiac scaffolds enriched with MSC-derived extracellular vesicles limit ventricular remodelling and exert local and systemic immunomodulation in a myocardial infarction porcine model

doi: 10.7150/thno.72289

Figure Lengend Snippet: Scaffold implantation modulates the systemic immune response post-MI, and cATMSC-EV promote a local effect. ( A-C ) Systemic analysis of the immune response post-MI. ( A ) The increase in the number of total peripheral blood mononuclear cells (PBMC) (left) and specifically in the number of lymphocytes (right) in whole blood that occurs 2 days after MI (red) is prevented by scaffold implantation. ( B ) The increase in the number of CCR2 + monocytes observed 30 days post-MI in untreated animals is blocked by scaffold implantation. ( C ) EV-Treated animals reduce the expression of CD73 in circulating monocytes 30 days from MI. Bars indicate mean ± SD and each data point corresponds to one animal (n = 4;7;8 respectively). Statistical significance was calculated with a paired Two-way ANOVA with Tukey's posthoc analysis. ( D-E ) Representative images ( D ) and quantification ( E ) of the number of infiltrating macrophages (CD163 + ; left), of which expressed CD73 (middle), and cells expressing CD73 (right) by immunohistofluorescence analysis at 30 days post-MI. Statistical significance was calculated with a One-way ANOVA with Tukey's posthoc analysis (Infarct core, left) and Student T-test (Scaffold, right). ( E ) CD163 (red), CD73 (green) and DAPI (blue).

Article Snippet: On one hand, 100 μL of whole blood were stained with the primary antibodies mouse anti-pig CD14-FITC (BioRad), anti-pig CD16-PE (BioRad), anti-human CCR2 (CD192)-PE-Vio770 (Miltenyi Biotech), rabbit anti-pig CD73 (Novus Biologicals) and mouse anti-pig CD163 (Novus Biologicals).

Techniques: Expressing, Immunohistofluorescence