Journal: Nature Communications

Article Title: Immunomodulating nano-adaptors potentiate antibody-based cancer immunotherapy

doi: 10.1038/s41467-021-21497-6

Figure Lengend Snippet: a imNA was constructed to enhance the antitumor activity of NK cells, and the following four groups were established: (1) IgG control; (2) Free αKLRG1 & αPDL1 ; (3) NP αKLRG1 & NP αPDL1 ; and (4) imNA αKLRG1 & αPDL1 . b Experimental protocol for the lung metastatic melanoma tumor model used in c – e : C57BL/6 mice were injected with 5.0 × 10 4 B16-F10 melanoma cells via the tail vein, and treatment started on the second day. The equivalent dose of αKLRG1 and αPDL1 was 1.5 mg/kg. c Images of lung tissues at 20 days post tumor cell infusion. d Calculation of tumor nodules on the lung tissues. Data are presented as means ± s.d. n = 3 or 6 biologically independent mice, each pot represents one mouse. e Representative H&E images of lung tissues. f imNA was constructed to enhance the antitumor activity of macrophages, and the following four groups were established: (1) IgG control; (2) Free αCSF1R & αCD47 ; (3) NP αCSF1R & NP αCD47 ; and (4) imNA αCSF1R & αCD47 . Representative flow cytometric analysis images ( g ) and relative quantification ( h ) of the phagocytosis of cancer cells by bone marrow-derived macrophages (BMDMs). CFSE-labeled B16-F10 cells were incubated with BMDMs for 4 h in the presence of IgG control, Free αCSF1R & αCD47 , NP αCSF1R & NP αCD47 or imNA αCSF1R & αCD47 , and then subjected to flow cytometric detection. Phagocytosis was quantified as the percentage of CFSE-positive BMDMs. Data are presented as means ± s.d. n = 3 biologically independent samples. i Experimental protocol for the B16-F10 melanoma model used in j , k , the equivalent dose of αCSF1R and αCD47 was 1.5 mg/kg, and the dose of IgG control was 3.0 mg/kg. Individual ( j ) and average ( k ) tumor growth curves of B16-F10 tumors in mice receiving different treatments. Data are presented as means ± s.d. n = 6 biologically independent mice. Statistical significance was calculated via one-way ANOVA with the Tukey post-hoc test. ** P < 0.01; *** P < 0.001; **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: The InVivo Plus anti-mouse PD1 antibody (Clone: 29 F.1A12), anti-mouse PDL1 antibody (Clone: 10 F.9G2), anti-mouse KLRG1 antibody (Clone: 2F1), anti-mouse CD47 antibody (Clone: MIAP410), anti-mouse CSF1R antibody (Clone: AFS98) and rat IgG2a isotype control (anti-trinitrophenol, Clone: 2A3) were obtained from Bio X Cell.

Techniques: Construct, Activity Assay, Control, Injection, Quantitative Proteomics, Derivative Assay, Labeling, Incubation

Journal: Cell reports

Article Title: Bone Morphogenic Proteins Are Immunoregulatory Cytokines Controlling FOXP3 + T reg Cells

doi: 10.1016/j.celrep.2020.108219

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti-Mouse Klrg-1-Biotin (2F1) , BD Bioscience , Cat# 550863, RRID:AB_393931.

Techniques: Recombinant, Magnetic Beads, Staining, Chromatin Immunoprecipitation, SYBR Green Assay, Knock-Out, Software

Journal: Cancer cell

Article Title: Lymphoma accelerates T cell and tissue aging

doi: 10.1016/j.ccell.2025.07.023

Figure Lengend Snippet: (A) Gating strategy and quantification of naive (N), effector (E), central memory (CM), and CD62L − CD44 lo CD4 + T cell populations ( n = 5). (B–E) Cell surface expression and representative histograms on CD4 + T cells ( n = 5): (B) CD39, (C) CD69, (D) KLRG1, and (E) PD-1. Line in flow plots represents where positivity was determined. (F–H) Intracellular expression and representative histograms of transcription factors in the indicated CD4 + T cells ( n = 3–4) (F) Tbet, (G) Tox, and (H) Foxp3. (I) Gating strategy for CD11b/CD27 NK cell maturation subtypes ( n = 3). (J) Cell surface expression and representative histogram of CXCR3 in NK cells ( n = 3/group). YC, YL, AC, AL are defined in . Data are mean ± SEM and are analyzed by a two-way ANOVA and Sidak’s test (A and I) or Student’s t test (B–H and J). Data are compiled from at least two independent experiments, and each dot represents an individual mouse. * p < 0.05 ** p < 0.01, and *** p < 0.001, **** p < 0.0001, n.s. = not significant. See also and .

Article Snippet: KLRG1 – APC , Tonbo , Cat # 20-5893; RRID:AB_2621607.

Techniques: Expressing

Journal: Cell reports

Article Title: Impact of regulated secretion on anti-parasitic CD8 T cell responses

doi: 10.1016/j.celrep.2014.04.031

Figure Lengend Snippet: C57BL/6 mice were immunized with irradiated T. gondii tachyzoites. Splenocytes were harvested from mice 2 wks post immunization and T. gondii-specific CD8 and CD4 T cell responses were measured by intracellular cytokine staining for IFN-γ. APCs +/- T. gondii were used as stimulators for ex-vivo and in vitro restimulations. Ex-vivo and in-vitro restimulation results are shown as A) Representative flow cytometry plots B and C) Plots depicting the expansion of B) CD8 T cells C) and CD4 T cells over the course of ten in-vitro restimulations. Data are representative of three experiments. D) LacZ response of sub-cloned CD8 hybridoma (BTg45Z) after an overnight culture with wild-type APCs +/- T. gondii or APCs blocked with antibodies against H-2Kb or H-2Db MHC I molecules + T. gondii. Data is representative of at least two independent experiments.

Article Snippet: Surface staining with anti-mouse CD4 (RM4-5), CD8α (53-6.7), IFN-γ (XMG1.2) (BD biosciences), anti-mouse KLRG-1 and IL7R (eBiosciences or BioLegend) antibodies was done as per manufacturer's instructions.

Techniques: Irradiation, Staining, Ex Vivo, In Vitro, Flow Cytometry, Clone Assay

Journal: Cell reports

Article Title: Impact of regulated secretion on anti-parasitic CD8 T cell responses

doi: 10.1016/j.celrep.2014.04.031

Figure Lengend Snippet: A-E) Mice were orally infected with 25-40 T. gondii cysts or F-H) immunized with irradiated 5×106 T. gondii, ME49 tachyzoites. Splenocytes and brain leukocytes were harvested from mice 3-4 wks after oral infection or 2 wks after immunization. T. gondii-specific T cell responses were measured by intracellular cytokine staining for IFN-γ or by staining with peptide-MHC tetramers. A) Representative flow cytometry plots of splenocytes from naïve and infected mice. Left panels show intracellular IFN-γ staining after in vitro restimulation with antigen presenting cells either, +/- T. gondii, or 1μM YAL9 peptide. Right panels show MHC class I H-2Db–YAL9 tetramer staining. B, D) Compiled data showing T. gondii-specific and H-2Db–YAL9-specific responses from B) spleen and D) brain of infected mice. Data are corrected for background based on responses by CD8 T cells towards uninfected APCs or APCs pulsed with irrelevant peptide. Each dot represents an individual mouse. C, E) Compiled data showing flow cytometry analysis of MHC class I H-2Db–YAL9 tetramer staining on C) splenocytes and E) brain leukocytes. Cells were also co-stained with CD8 antibody. F) Representative flow cytometry plots of splenocytes from naïve and immunized mice. Left panels show intracellular IFN-γ staining after in vitro restimulation with antigen presenting cells either, +/- T. gondii, or 1μM YAL9 peptide. Right panels show MHC class I H-2Db–YAL9 tetramer staining. G) Compiled data showing splenic T. gondii-specific and H-2Db–YAL9-specific responses from immunized mice. Data are corrected for background based on responses by CD8 T cells towards uninfected APCs or APCs pulsed with irrelevant peptide. Each dot represents an individual mouse. H) Compiled data showing MHC class I H-2Db–YAL9 tetramer staining on splenocytes from immunized mice. Cells were also co-stained with CD8 antibody. Data are representative of at least three independent experiments. See also Figure S3.

Article Snippet: Surface staining with anti-mouse CD4 (RM4-5), CD8α (53-6.7), IFN-γ (XMG1.2) (BD biosciences), anti-mouse KLRG-1 and IL7R (eBiosciences or BioLegend) antibodies was done as per manufacturer's instructions.

Techniques: Infection, Irradiation, Staining, Flow Cytometry, In Vitro

Journal: Cell reports

Article Title: Impact of regulated secretion on anti-parasitic CD8 T cell responses

doi: 10.1016/j.celrep.2014.04.031

Figure Lengend Snippet: A) Schematic depicting distinct modes of secretion for the dense granule protein GRA6 (depicted in red) (Holtappels et al., 2008), versus rhoptry protein ROP5 (depicted in blue). Also shown are possible routes for antigen processing and presentation via MHC-I. T. gondii injects rhoptry proteins directly into the host cell during invasion. ROP5 associates with the cytosolic face of parasitophorous vacuole membrane (PVM) following secretion. Parasites constitutively secrete dense granule proteins into the PV lumen of invaded host cells. Proteolytic processing of parasite secretory proteins could take place in the host cells' cytosol by the proteasome, and further trimming by ERAAP may occur after TAP-mediated transport of peptides into the ER. Processed peptides are loaded onto MHC class I molecules to be transported to the cell surface for recognition by a CD8 T cell (not depicted). B) Left hand diagrams show wild type ROP5 and GRA6 genes indicating the location of their antigenic epitopes (YAL9 and HF10) and placement of HA and FLAG epitope tags. Right hand diagrams show the transgenic constructs used to retarget expression of the epitopes to different parasite secretory compartments. GRA6-YAL9 has the HF10 epitope of the GRA6 gene replaced by YAL9. An additional 10 amino acids from ROP5 flanking the YAL9 epitope were included to allow for efficient processing (not depicted). GRA6-ROP5 contains the GRA6 promoter and signal sequence fused to the C-terminal portion of the ROP5IIC gene including the YAL9 epitope. ROP5-HF10 contains the HF10 epitope from GRA6 fused to the C-terminus of ROP5IIIC gene. All constructs were introduced as transgenes into the parental type III strain, CTG, which harbors allelic forms of ROP5 and GRA6 that lack the T cell stimulatory epitopes. C) BMDCs from C57BL/6 (H-2b) or B10.D2 (H-2d) mice were infected in vitro with irradiated transgenic T. gondii tachyzoites at varying MOI and BTg45Z and CTgEZ.4 lacZ response was measured after an overnight stimulation with BMDCs +/- T. gondii. D) C57BL/6 mice (H-2b) were infected i.p. with 105 transgenic T. gondii tachyzoites. Splenocytes or brain leukocytes were harvested from mice 3-4 wks post infection and T. gondii-specific T cell responses were measured by staining with tetramers. Compiled data showing MHC class I H-2Db–YAL9 tetramer staining on gated CD8+ B220-splenocytes (left panel) and brain leukocytes (right panel). F) Compiled data showing splenic (left panel) and brain (right panel) T. gondii-specific and H-2Db-YAL9-specific CD8 T cell responses from infected mice as measured by intracellular cytokine staining for IFN-γ. Data are corrected for background based on responses by T cells towards uninfected APCs or APCs pulsed with irrelevant peptide. G) Plots depicting the ratio of T. gondii-specific CD4 to CD8 T cell response from spleen (left panel) or brain (right panel) of infected mice. H) Mice were immunized with irradiated 5×106 transgenic T. gondii tachyzoites. Splenocytes were harvested from mice 2 wks post immunization and T. gondii-specific T cell responses were measured using intracellular IFN-γ staining for. Compiled data showing splenic T. gondii and YAL9-specific CD8 T cell responses from immunized mice. Data are corrected for background based on responses by T cells towards uninfected APCs or APCs pulsed with irrelevant peptide. (I) Compiled data showing MHC class I H-2Db–YAL9 tetramer staining on splenocytes from immunized mice. Cells were also co-stained with CD8 antibody. Each bar represents an average from six mice. Each dot represents an individual mouse. Data are pooled from two independent experiments. (J) B6×B6.C (H-2b/d) mice were infected with the indicated transgenic T. gondii tachyzoites. Splenocytes or brain leukocytes were harvested from mice 21 days post infection and GRA6-specific T cell responses were measured by staining with H-2Ld-HF10 tetramers. Compiled tetramer staining data on gated CD8+ B220- splenocytes (left panel) and CD8+ brain leukocytes (right panel). (*=p<0.05, **=p<0.01, ***=p<0.001,****=p<0.0001). See also Figure S4 and S5.

Article Snippet: Surface staining with anti-mouse CD4 (RM4-5), CD8α (53-6.7), IFN-γ (XMG1.2) (BD biosciences), anti-mouse KLRG-1 and IL7R (eBiosciences or BioLegend) antibodies was done as per manufacturer's instructions.

Techniques: FLAG-tag, Transgenic Assay, Construct, Expressing, Sequencing, Infection, In Vitro, Irradiation, Staining

Journal: Cell reports

Article Title: Impact of regulated secretion on anti-parasitic CD8 T cell responses

doi: 10.1016/j.celrep.2014.04.031

Figure Lengend Snippet: A) Mice were immunized with LPS activated BMDCs pulsed with YAL9 or control peptide (WI9), boosted after 2 wks, and after 3 wks challenged with 5×105 live transgenic T. gondii tachyzoites i.p. (either CTG+ROP5 or CTG+GRA6-ROP5). B) Kaplan-Meier survival curves between the four groups of mice. C-F) Mice that survived the acute phase of infection were sacrificed at 7 wks post challenge. C) MHC class I H-2Db–YAL9 tetramer staining on brain leukocytes from surviving mice. Cells were also co-stained with CD8 antibody. D) CD8 T cells or E) ratio of T. gondii-specific CD4 to CD8 T cells in the brain as measured by ICCS for IFN-γ using flow cytometry after ex-vivo restimulation with T. gondii-infected APCs or peptide-pulsed APCs. Data are background corrected based on the values from uninfected APCs or APC pulsed with irrelevant peptide. F) Number of cysts in the brain as measured by staining a portion of the brain with fluorescent lectin to detect the cysts. Data is pooled from two representative experiments with at least 5 mice per condition in each experiment. (*=p<0.05, **=p<0.01, ***=p<0.001, ****=p<0.0001, NS = not significant). See also Figure S6.

Article Snippet: Surface staining with anti-mouse CD4 (RM4-5), CD8α (53-6.7), IFN-γ (XMG1.2) (BD biosciences), anti-mouse KLRG-1 and IL7R (eBiosciences or BioLegend) antibodies was done as per manufacturer's instructions.

Techniques: Transgenic Assay, Infection, Staining, Flow Cytometry, Ex Vivo