Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Synergistic blockade of SHP-2 and A2AR signal pathways with targeted nanoparticles restores anti-tumor immunity of CD8+ T cells.

doi: 10.1016/j.jconrel.2025.113889

Figure Lengend Snippet: Fig. 1. Characteristics of dual-inhibitor loaded SCNPs/αCD8 nanoparticles. (a) SEM images of SCNPs and (b) SCNPs/αCD8 showing an average size of ~230 nm and having a spherical morphology. (c) The hydrodynamic size distribution of SCNPs (red) and SCNPs/αCD8 (black) measured via dynamic light scattering (DLS). (d) Release kinetics profiles of SHP099 and CPI-444 from SCNPs/αCD8 at physiological pH (7.4) in PBS and (e) pH 6.0 PBS containing 10 % serum. (Mean ± SD; n = 3 three independent experiments). (f) The colloidal stability of SCNPs/αCD8 in terms of the change in the average particle size and zeta potential in pH 7.4 PBS and pH 7.4 PBS containing 10 % serum, measured by DLS over a 3-day period at 4 ◦C. (Mean ± SD; n = 3 three independent experiments). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Human anti-CD8 antibody (denoted αCD8), isotype IgG and mouse anti-CD8 antibody (denoted mαCD8) were purchased from R&D Systems.

Techniques: Zeta Potential Analyzer

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Synergistic blockade of SHP-2 and A2AR signal pathways with targeted nanoparticles restores anti-tumor immunity of CD8+ T cells.

doi: 10.1016/j.jconrel.2025.113889

Figure Lengend Snippet: Fig. 2. Targeted delivery inhibitors and biological activity of SCNP/αCD8 nanoparticles. (a) The fluorescence microscope images for cellular internalization of C6- labeled nanoparticles. Scale bar: 50 μm. (b) Quantitative analysis of cellular internalization of dye-labeled nanoparticles by flow cytometry. The relative amount of SHP099 (c) and CPI-444 (d) delivered into CD8+T cell after 24 h incubation as quantified by LS-MS-MS. (e) Western blot analysis of the expression of SHP2, pSHP2, tPI3K, pPI3K, tERK and pERK in CD8+ T cell after different treatments. β-actin was used as the internal control. (f-h) Quantification of the expression of pSHP2/tSHP2 (f), pPI3K/tPI3K (g), and pERK/tERK (h) in CD8+ T cells. Data shown are mean ± SD (n = 3, three independent experiments), **p < 0.01, ****p < 0.0001.

Article Snippet: Human anti-CD8 antibody (denoted αCD8), isotype IgG and mouse anti-CD8 antibody (denoted mαCD8) were purchased from R&D Systems.

Techniques: Activity Assay, Fluorescence, Microscopy, Labeling, Flow Cytometry, Incubation, Tandem Mass Spectroscopy, Western Blot, Expressing, Control

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Synergistic blockade of SHP-2 and A2AR signal pathways with targeted nanoparticles restores anti-tumor immunity of CD8+ T cells.

doi: 10.1016/j.jconrel.2025.113889

Figure Lengend Snippet: Fig. 3. Impact of SCNPs/αCD8 nanoparticles on CD8+T cell activation and function. (a) Fluorescence signal dilution of CD8+ T cells labeled with CFSE following activation with αCD3/CD28 in the presence of different nanoparticle formulations. (b) Fold expansion of CD8+ T cell population after incubation with different nanoparticle formulations together with αCD3/CD28. (c-d) Expression of CD25(c) and CD69(d) in CD8+ T cells incubated with αCD3/CD28 and different nano particle formulations. (e-f) IL-2 (e) and IFN-γ (f) secretion by CD8+ T cells after different treatments. (g-h) Flow cytometry data of CD8+ T cells positive with granzyme B (g) and perforin (h). (i) Tumor cell killing percent detected by LDH release. Data are presented as mean ± SD of three independent experiments. **p < 0.01.

Article Snippet: Human anti-CD8 antibody (denoted αCD8), isotype IgG and mouse anti-CD8 antibody (denoted mαCD8) were purchased from R&D Systems.

Techniques: Activation Assay, Fluorescence, Labeling, Incubation, Expressing, Flow Cytometry

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Synergistic blockade of SHP-2 and A2AR signal pathways with targeted nanoparticles restores anti-tumor immunity of CD8+ T cells.

doi: 10.1016/j.jconrel.2025.113889

Figure Lengend Snippet: Fig. 4. SCNP/αCD8 restored T-cell activity in the presence of immune suppression. (a) PD-1 expression on human CD8+ T cells following activation with αCD3/CD28 beads complex. (b) A2AR expression on CD8+ T cells following activation with anti-CD3/CD28 complex. Flow cytometry assay of T cells positive with CD107a (c) and CD69 (d) when αCD3/CD28 activated CD8+ T cell co-cultured with CGS-21680 and peptide (10 mM) in the present or absence dual-drug loaded untargeted, isotype and targeted nanoparticles. Data are shown as mean ± SD, n = 3. ****p < 0.0001.

Article Snippet: Human anti-CD8 antibody (denoted αCD8), isotype IgG and mouse anti-CD8 antibody (denoted mαCD8) were purchased from R&D Systems.

Techniques: Activity Assay, Expressing, Activation Assay, Flow Cytometry, Cell Culture

Journal: Journal of controlled release : official journal of the Controlled Release Society

Article Title: Synergistic blockade of SHP-2 and A2AR signal pathways with targeted nanoparticles restores anti-tumor immunity of CD8+ T cells.

doi: 10.1016/j.jconrel.2025.113889

Figure Lengend Snippet: Fig. 5. Therapeutic efficacy of SCNP/mαCD8 in C57BL/6 Hepa1–6 tumor-bearing mice. (a) Schematic schedule of SCNP/mαCD8 treatments in mouse model (n = 7/ group) with PBS, SHP099 + CPI-444, SCNP and SCNP/mαCD8 (20 mg/kg for each drug). (b) The average tumor volume change vs treatment time; and individual tumor growth profiles in each group. The photo (c) and weight (d) of tumor tissues collected at day 18. Data are mean ± SD (n = 7 per group). (e) Multiplex immunofluorescence staining of CD3+CD8+ T cells infiltrating into tumor tissues of the treated mice. Magnification:100×, bar: 50 μm (up panel) and 100 μm (bottom panel). (f) Colonies and (e) the number of IFN-γ producing CD8+ T cells in tumor tissues of the treated mice by ELISPOT assay. Data are shown as mean ± SD (n = 3).

Article Snippet: Human anti-CD8 antibody (denoted αCD8), isotype IgG and mouse anti-CD8 antibody (denoted mαCD8) were purchased from R&D Systems.

Techniques: Drug discovery, Multiplex Assay, Immunofluorescence, Staining, Enzyme-linked Immunospot

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus.

doi: 10.4049/jimmunol.177.10.7423

Figure Lengend Snippet: FIGURE 2. Connective tissue disease ar- rays. Ordered Ag arrays were produced using a robotic microarrayer to attach lupus-associated Ags and Abs directed against mouse IgG (-IgG) to poly-L-lysine-coated microscope slides. Individual arrays were probed with di- luted mouse sera. The positions of select au- toantigens and control features are indicated. A, An array probed with normal control C57BL/6 mouse serum showed no autoanti- body reactivity. B, Representative images of arrays probed with sera from two 20-wk-old F9 H2k/k MRL/lpr mice. C, Representative im- ages of arrays probed with sera from two F9 H2b/b MRL/lpr mice. D, Quantitative analysis of A–C. Results are expressed as normalized DFU (see Materials and Methods).

Article Snippet: Serum dilutions were then incubated in the coated wells for 1 h, and bound Abs were detected with HRP-conjugated anti-mouse IgM, IgG1, IgG2a, IgG2b, or IgG3 (Southern Biotechnology Associates) and developed with TMB (Sigma-Aldrich), 0.1 M citrate buffer (pH 4.0), and 0.015% H2O2.

Techniques: Produced, Microscopy, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus.

doi: 10.4049/jimmunol.177.10.7423

Figure Lengend Snippet: FIGURE 4. Serum autoantibodies in MRL/lpr mice. Serum IgG anti-dsDNA (A), IgG anti-GA (C), IgG3 anti-mouse IgG2a RF (D), and IgG anti-Sm (E) levels in the F9 H2k/k, H2b/k, and H2b/b MRL/lpr mice were measured by ELISA. n 12–16 in each group at 12 wk of age. The number of the mice in each group decreased with aging due to mortality. Twenty-week-old mice sera from each group were used for IgG subclass analysis of anti-dsDNA (B) and anti-Sm (F) Abs. , p 0.05; , p 0.01; , p 0.001.

Article Snippet: Serum dilutions were then incubated in the coated wells for 1 h, and bound Abs were detected with HRP-conjugated anti-mouse IgM, IgG1, IgG2a, IgG2b, or IgG3 (Southern Biotechnology Associates) and developed with TMB (Sigma-Aldrich), 0.1 M citrate buffer (pH 4.0), and 0.015% H2O2.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus.

doi: 10.4049/jimmunol.177.10.7423

Figure Lengend Snippet: FIGURE 5. Serum Ig levels and Ab-producing cells in MRL/lpr mice. A, Serum concentrations of IgM (upper panel), IgG2a (middle panel), and IgG3 (lower panel) in the 129/Sv strain-derived F9 H2k/k, H2b/k, and H2b/b MRL/lpr mice were determined by ELISA using standards of known concentration. Eleven of 16 (68.8%) of the H2b/b MRL/lpr mice had undetectable serum IgG3 levels. n 12–16 mice in each group at 12 wk of age. The number of the mice in each group decreased with aging due to mortality. , p 0.05; , p 0.01; , p 0.001. B, Populations of Ig isotype producing cells in the spleens of 32-wk-old F9 H2k/k, H2b/k, and H2b/b MRL/lpr mice. The F9 H2 MRL/lpr mice were sacrificed at 32 wk, and spleen cells were analyzed by ELISPOT assay. n 7 in the H2k/k, 5 in the H2b/k, and 10 in the H2b/b MRL/lpr mice for IgG isotype analysis. n 3, 3, and 5 in the H2k/k, H2b/k, and H2b/b MRL/lpr mice for IgM-producing cell analysis, respectively. , p 0.01; , p 0.001. C, Serum concentrations of IgG3 in 12-wk-old 129/Sv, B6, or BALB/c strain-derived F3 H2k/k, H2b/k, H2b/b, H2d/k, and H2d/d MRL/lpr mice were determined by ELISA using standards of murine IgG3 of known concentration. , p 0.05; , p 0.001.

Article Snippet: Serum dilutions were then incubated in the coated wells for 1 h, and bound Abs were detected with HRP-conjugated anti-mouse IgM, IgG1, IgG2a, IgG2b, or IgG3 (Southern Biotechnology Associates) and developed with TMB (Sigma-Aldrich), 0.1 M citrate buffer (pH 4.0), and 0.015% H2O2.

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Enzyme-linked Immunospot, Cell Analysis

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus.

doi: 10.4049/jimmunol.177.10.7423

Figure Lengend Snippet: FIGURE 6. Renal disease assessment in MRL/lpr mice. A, Urinary albumin excretion levels in the H2k/k, IgG3 H2b/b, and IgG3 H2b/b MRL/lpr mice. Data presented are the mean 24-h albumin excretion (mg/mouse/day) SEM in each group. n 11, 8, and 11 in the H2k/k, IgG3 H2b/b, and IgG3 H2b/b MRL/lpr mice, respectively. B, Albuminuria development (Kaplan-Meier curve) in the H2k/k, IgG3 H2b/b, and IgG3 H2b/b

Article Snippet: Serum dilutions were then incubated in the coated wells for 1 h, and bound Abs were detected with HRP-conjugated anti-mouse IgM, IgG1, IgG2a, IgG2b, or IgG3 (Southern Biotechnology Associates) and developed with TMB (Sigma-Aldrich), 0.1 M citrate buffer (pH 4.0), and 0.015% H2O2.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus.

doi: 10.4049/jimmunol.177.10.7423

Figure Lengend Snippet: FIGURE 7. Histological, immunofluorescence, and EM studies of kidneys from MRL/lpr mice. A, Representative sections of kidneys from MRL/lpr mice differing in H2 and IgG3 production. Total IgG, IgG3, and C3 glomerular deposition as assessed by immunofluores- cence are depicted in the left three panels. Proliferative glomerular disease and inflammation are demonstrated in H&E-stained paraffin sec- tions. B–D, EM analysis of kidneys from F9 H2k/k (B), IgG3 H2b/b (C), and IgG3 H2b/b (D) MRL/lpr mice. Both the H2k/k and IgG3 H2b/b

Article Snippet: Serum dilutions were then incubated in the coated wells for 1 h, and bound Abs were detected with HRP-conjugated anti-mouse IgM, IgG1, IgG2a, IgG2b, or IgG3 (Southern Biotechnology Associates) and developed with TMB (Sigma-Aldrich), 0.1 M citrate buffer (pH 4.0), and 0.015% H2O2.

Techniques: Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus.

doi: 10.4049/jimmunol.177.10.7423

Figure Lengend Snippet: FIGURE 8. Survival of MRL/lpr mice differing in H2 and IgG3 pro- duction. Mortality was observed until the time of sacrifice (32 wk). There were no differences in survival between the groups. n 13–16 in each group.

Article Snippet: Serum dilutions were then incubated in the coated wells for 1 h, and bound Abs were detected with HRP-conjugated anti-mouse IgM, IgG1, IgG2a, IgG2b, or IgG3 (Southern Biotechnology Associates) and developed with TMB (Sigma-Aldrich), 0.1 M citrate buffer (pH 4.0), and 0.015% H2O2.

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus.

doi: 10.4049/jimmunol.177.10.7423

Figure Lengend Snippet: FIGURE 2. Connective tissue disease ar- rays. Ordered Ag arrays were produced using a robotic microarrayer to attach lupus-associated Ags and Abs directed against mouse IgG (-IgG) to poly-L-lysine-coated microscope slides. Individual arrays were probed with di- luted mouse sera. The positions of select au- toantigens and control features are indicated. A, An array probed with normal control C57BL/6 mouse serum showed no autoanti- body reactivity. B, Representative images of arrays probed with sera from two 20-wk-old F9 H2k/k MRL/lpr mice. C, Representative im- ages of arrays probed with sera from two F9 H2b/b MRL/lpr mice. D, Quantitative analysis of A–C. Results are expressed as normalized DFU (see Materials and Methods).

Article Snippet: The assay was then performed as described above with HRP-conjugated goat anti-mouse IgG3 (Southern Biotechnology Associates).

Techniques: Produced, Microscopy, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus.

doi: 10.4049/jimmunol.177.10.7423

Figure Lengend Snippet: FIGURE 4. Serum autoantibodies in MRL/lpr mice. Serum IgG anti-dsDNA (A), IgG anti-GA (C), IgG3 anti-mouse IgG2a RF (D), and IgG anti-Sm (E) levels in the F9 H2k/k, H2b/k, and H2b/b MRL/lpr mice were measured by ELISA. n 12–16 in each group at 12 wk of age. The number of the mice in each group decreased with aging due to mortality. Twenty-week-old mice sera from each group were used for IgG subclass analysis of anti-dsDNA (B) and anti-Sm (F) Abs. , p 0.05; , p 0.01; , p 0.001.

Article Snippet: The assay was then performed as described above with HRP-conjugated goat anti-mouse IgG3 (Southern Biotechnology Associates).

Techniques: Enzyme-linked Immunosorbent Assay

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus.

doi: 10.4049/jimmunol.177.10.7423

Figure Lengend Snippet: FIGURE 5. Serum Ig levels and Ab-producing cells in MRL/lpr mice. A, Serum concentrations of IgM (upper panel), IgG2a (middle panel), and IgG3 (lower panel) in the 129/Sv strain-derived F9 H2k/k, H2b/k, and H2b/b MRL/lpr mice were determined by ELISA using standards of known concentration. Eleven of 16 (68.8%) of the H2b/b MRL/lpr mice had undetectable serum IgG3 levels. n 12–16 mice in each group at 12 wk of age. The number of the mice in each group decreased with aging due to mortality. , p 0.05; , p 0.01; , p 0.001. B, Populations of Ig isotype producing cells in the spleens of 32-wk-old F9 H2k/k, H2b/k, and H2b/b MRL/lpr mice. The F9 H2 MRL/lpr mice were sacrificed at 32 wk, and spleen cells were analyzed by ELISPOT assay. n 7 in the H2k/k, 5 in the H2b/k, and 10 in the H2b/b MRL/lpr mice for IgG isotype analysis. n 3, 3, and 5 in the H2k/k, H2b/k, and H2b/b MRL/lpr mice for IgM-producing cell analysis, respectively. , p 0.01; , p 0.001. C, Serum concentrations of IgG3 in 12-wk-old 129/Sv, B6, or BALB/c strain-derived F3 H2k/k, H2b/k, H2b/b, H2d/k, and H2d/d MRL/lpr mice were determined by ELISA using standards of murine IgG3 of known concentration. , p 0.05; , p 0.001.

Article Snippet: The assay was then performed as described above with HRP-conjugated goat anti-mouse IgG3 (Southern Biotechnology Associates).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay, Enzyme-linked Immunospot, Cell Analysis

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus.

doi: 10.4049/jimmunol.177.10.7423

Figure Lengend Snippet: FIGURE 6. Renal disease assessment in MRL/lpr mice. A, Urinary albumin excretion levels in the H2k/k, IgG3 H2b/b, and IgG3 H2b/b MRL/lpr mice. Data presented are the mean 24-h albumin excretion (mg/mouse/day) SEM in each group. n 11, 8, and 11 in the H2k/k, IgG3 H2b/b, and IgG3 H2b/b MRL/lpr mice, respectively. B, Albuminuria development (Kaplan-Meier curve) in the H2k/k, IgG3 H2b/b, and IgG3 H2b/b

Article Snippet: The assay was then performed as described above with HRP-conjugated goat anti-mouse IgG3 (Southern Biotechnology Associates).

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus.

doi: 10.4049/jimmunol.177.10.7423

Figure Lengend Snippet: FIGURE 7. Histological, immunofluorescence, and EM studies of kidneys from MRL/lpr mice. A, Representative sections of kidneys from MRL/lpr mice differing in H2 and IgG3 production. Total IgG, IgG3, and C3 glomerular deposition as assessed by immunofluores- cence are depicted in the left three panels. Proliferative glomerular disease and inflammation are demonstrated in H&E-stained paraffin sec- tions. B–D, EM analysis of kidneys from F9 H2k/k (B), IgG3 H2b/b (C), and IgG3 H2b/b (D) MRL/lpr mice. Both the H2k/k and IgG3 H2b/b

Article Snippet: The assay was then performed as described above with HRP-conjugated goat anti-mouse IgG3 (Southern Biotechnology Associates).

Techniques: Staining

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Role of MHC-linked genes in autoantigen selection and renal disease in a murine model of systemic lupus erythematosus.

doi: 10.4049/jimmunol.177.10.7423

Figure Lengend Snippet: FIGURE 8. Survival of MRL/lpr mice differing in H2 and IgG3 pro- duction. Mortality was observed until the time of sacrifice (32 wk). There were no differences in survival between the groups. n 13–16 in each group.

Article Snippet: The assay was then performed as described above with HRP-conjugated goat anti-mouse IgG3 (Southern Biotechnology Associates).

Techniques:

Journal: Human Vaccines & Immunotherapeutics

Article Title: Identification of immunogenic MAGED4B peptides for vaccine development in oral cancer immunotherapy

doi: 10.4161/hv.29226

Figure Lengend Snippet: MAGED4B-specific peptides are immunogenic. (A) T cells secreting Granzyme B and IFN-γ were detected in ex vivo ELISPOT assays with MAGED4B peptides; HLA-A2 restricted FluM-derived peptide was used as a positive control, culture medium alone without peptide was used as negative control; (B) The production of both Granzyme B and IFN-γ spots (cytokine secreting cells) was significantly elevated in young patients as compared with old OSCC patients; the mean age of all patients (60 yr) was used as a cutoff point to distinguish the old and young individuals.

Article Snippet: The spots present in each well were scanned using the CTL ELISPOT analyzer and counted using the ImmunoSpot Professional Software (CTL Analyzers LLC).

Techniques: Ex Vivo, Enzyme-linked Immunospot, Derivative Assay, Positive Control, Negative Control