Journal: Pharmaceutics

Article Title: Surface Functionalization of Silica Nanoparticles: Strategies to Optimize the Immune-Activating Profile of Carrier Platforms

doi: 10.3390/pharmaceutics14051103

Figure Lengend Snippet: Co-stimulatory molecule expression by stimulated moDCs with functionalized SiNPs and Bet. ( A ) CD40, ( B ) CD86, ( C ) CD83, ( D ) HLA-DR, and ( E ) CD80 were analyzed. LPS was used as the positive control and a statistical significance was observed in comparison to the cell control indicating a successful stimulation. All particles and conjugates were compared to Bet v 1 for statistical analysis ( n = 6 individual donors) (* p ≤ 0.05; ** p ≤ 0.01; **** p ≤ 0.0001).

Article Snippet: The cells were then collected and stained for α-HLA-DR APC (Invitrogen, Waltham, MA, USA), Fixable Viability Dye eFluor506 (eBioscience, Waltham, MA, USA), α-CD1a BV421 (Biolegend, San Diego, CA, USA), α-CD86 PE (eBioscience), α-CD40 FITC (Biolegend), and α-CD83 PE-Cy™7 (BD Bioscience, Heidelberg, Germany).

Techniques: Expressing, Positive Control

Journal: Frontiers in Immunology

Article Title: Immune regulatory adjuvant approach to mitigate subcutaneous immunogenicity of monoclonal antibodies

doi: 10.3389/fimmu.2024.1496169

Figure Lengend Snippet: Impact of OPLS on RTX- and ADM-mediated T-cell activation when administered subcutaneously in mice. CD4 + T cells in the draining lymph nodes (DLN) of mice at the study terminal were stained for the activation marker CD44 and classical immune checkpoints CLTA-4 and PD-1. Supplementary Figure 3 is the flow cytometry gating strategy. Frequency (%) of (A) CD44 ++ and (B) CTLA-4 + PD-1 + cells out of live CD3 + CD4 + T cells in DLN of mice administered RTX + rHuPH20 in vehicle, 25 mM OPLS, or 100 mM OPLS or (C) in DLN of mice administered ADM in vehicle, 25 mM OPLS, or 50 mM OPLS. Dots represent individual mice and bars are mean ± SD. Statistical significance was determined by Student’s unpaired t-test (one-tailed). *p<0.05, **p<0.01.

Article Snippet: Where indicated, splenocytes were surface stained with anti-mouse CD4 APC-Cy7 (GK1.5), CD3 FITC (17A2), LAG-3 APC (C9B7W), CD49 PE (HMA2), and LAP PerCP-Cy5.5 (TW7-16B4) and stained intracellularly with anti-mouse FoxP3 Alexa Fluor 700 (MF-14) using a fixation/permeabilization kit (Tonbo Biosciences).

Techniques: Activation Assay, Staining, Marker, Flow Cytometry, One-tailed Test

Journal: Frontiers in Immunology

Article Title: Immune regulatory adjuvant approach to mitigate subcutaneous immunogenicity of monoclonal antibodies

doi: 10.3389/fimmu.2024.1496169

Figure Lengend Snippet: Co-administration with OPLS promotes differentiation of regulatory T cells in vivo and in vitro . In the ADM study ( Figure 1 ), mouse splenocytes were collected at the terminal endpoint and analyzed for Tr1 phenotype. Supplementary Figure 4 is the flow cytometry gating strategy. (A) Frequency (%) of LAG-3 + CD49b + FoxP3 - CD4 + Tr1 cells in the spleen. Dots represent individual mice and bars are mean ± SD. Statistical significance was determined by Student’s unpaired t-test (one-tailed). *p<0.05. (B, C) Naïve immature SW mouse BMDCs were cultured for 24 h with media, VitD3/Dex, OVA (a model antigen), and OVA + 25 mM or 50 mM OPLS. Splenocytes from SW mice (n=2) subcutaneously immunized with OVA (2 μg/100 μL) once weekly were collected 4 days after the second dose. Isolated CD4 + T cells were co-cultured with treated BMDCs at a ratio of 1:5 BMDC, CD4 + T cell for 72 h. Frequency (%) (B) LAG-3 + CD49 + Tr1 and (C) LAP + of CD4 + FoxP3 - T cells. Error bars are mean ± SD of triplicate wells.

Article Snippet: Where indicated, splenocytes were surface stained with anti-mouse CD4 APC-Cy7 (GK1.5), CD3 FITC (17A2), LAG-3 APC (C9B7W), CD49 PE (HMA2), and LAP PerCP-Cy5.5 (TW7-16B4) and stained intracellularly with anti-mouse FoxP3 Alexa Fluor 700 (MF-14) using a fixation/permeabilization kit (Tonbo Biosciences).

Techniques: In Vivo, In Vitro, Flow Cytometry, One-tailed Test, Cell Culture, Isolation

Journal: Frontiers in Immunology

Article Title: Immune regulatory adjuvant approach to mitigate subcutaneous immunogenicity of monoclonal antibodies

doi: 10.3389/fimmu.2024.1496169

Figure Lengend Snippet: OPLS does not impact immunocompetence of NHP and mice. CD-1 mice received daily SC doses of vehicle (n=6), 20 mg/kg CYP (n=3), or OPLS (n=6/group) at 18-450 mM for 28 days. NHP (rhesus macaques) (n=3) received 21 daily SC doses of 54 mM OPLS. (A, B) Frequencies of lymphocyte populations—CD19 + B cells, CD3 + T cells, CD3 + CD4 + T cells, and CD3 + CD8 + T cells—in (A) spleens of mice on day 28 and (B) peripheral blood of NHP on day 0 (baseline), 8, 18, 22, and 35. Supplementary Figure 5 is the gating strategy. (C) Anti-KLH IgM and (D) IgG titers (log 2 ) in plasma of mice on day 28 following a single IV dose of KLH (2 mg) on day 15. (E) Anti-KLH IgM and IgG titers (log 2 ) in plasma of NHP on day 18 and 22, respectively, following a single IM dose of KLH (10 mg) on day 8. Each dot represents an individual animal and all bars are mean ± SD. (F-K) Microscopic images of H&E-stained spleens from mice treated with (F) vehicle or (G) 18 mM, (H) 45 mM, (I) 90 mM, (J) 225 mM, and (K) 450 mM OPLS. Statistical significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test. ****p<0.0001.

Article Snippet: Where indicated, splenocytes were surface stained with anti-mouse CD4 APC-Cy7 (GK1.5), CD3 FITC (17A2), LAG-3 APC (C9B7W), CD49 PE (HMA2), and LAP PerCP-Cy5.5 (TW7-16B4) and stained intracellularly with anti-mouse FoxP3 Alexa Fluor 700 (MF-14) using a fixation/permeabilization kit (Tonbo Biosciences).

Techniques: Clinical Proteomics, Staining

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet: Single-Cell Survey Reveals Heterogeneity of cDC2s with Two Subsets Delineated by Expression of T-Bet (A) Representative contour plot showing gating strategy for splenic DCs in Tbx21 RFP-Cre mice. DCs defined as Lin(CD3,CD19,CD49b,Siglec-F) – Ly6C – CD64 – CD11c + MHCII + . (B) Frequency of T-bet + cDC2s across tissues. Each circle represents one mouse. In the peripheral and mesenteric LN (PLN and MLN), migratory DCs were defined as MHCII hi CD11c int and resident DCs as MHCII int CD11c hi . Error bars represent mean ± SEM. (C) Analysis of RFP + and YFP + splenic cDC2s from Tbx21 RFP-CreERT2 Rosa26 YFP mice, 3 days post tamoxifen gavage. (D) Percent RFP + and YFP + of cDC2 cells. Percent RFP + of YFP + cDC2s at indicated time points post tamoxifen gavage (right). Error bars represent mean ± SEM; n = 3–4 mice per time point. (E) t-SNE embedding of 4,464 DCs. Colors indicate unsupervised clustering by Phenograph (left panel) or classification based on expression of canonical markers (right panel). (F) Expression of canonical DC markers across the transcriptionally defined DC clusters from (E). (G) Proportion of T-bet (RFP + ) cells in each cell cluster identified in (D). (H) Violin plot showing expression of the cell-cycle signature across the DC clusters from (E). (I) Similarity of bulk T-bet – cDC2s, T-bet + cDC2, and cDC1 transcriptomes to the reference single-cell DC clusters (E). Colors represent the correlation coefficient between the cell population identified in the row label and the DC cluster identified by the column label. See also and .

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Expressing

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet: Single-Cell Survey Reveals Heterogeneity of cDC2s, Related to Figure 1 A. Representative histogram showing expression of T-bet (RFP) in splenic cells from Tbx21 RFP-cre mice. (B). Expression of T-bet in CD11b + XCR1 + DCs from the intestinal lamina propria. Data representative of > 5 independent experiments, with at least 3 mice per experiment. (C). Expression of T-bet in splenic myeloid cells. Cells were defined as: (i) Ly-6C hi monocytes (Lin – Ly6C + Ly6G – CD11b + CX3CR1 + ); neutrophils (Lin – Ly6C + Ly6G + ); macrophages (Lin – CD64 + Ly6C – ). Lineages (Lin) were defined as: CD3e, CD90.2, CD19, CD49b and Siglec F. Each circle represents an individual mouse, error bars represent mean ± SEM. (D). Left: Gating strategy for single-cell sorting. DCs were defined as Lin(CD3, CD19, CD90) – Ly6C – CD64 – CD11c + MHCII + . Two populations were sampled: RFP + DCs and RFP – DCs (encompassing XCR1 + cDC1s, CD11b + RFP – and CD11b – XCR1 – DCs). Right: Post-sort purity of RFP + and RFP – cells. Contaminating population of Ly6C + cells identifiable on post-sort purity (lower panel). (E). Similarity of splenic CD11c + MHCII + cells to reference myeloid cells (ImmGen Consortium) Colors represent the Pearson correlation between the mean gene expression from the dendritic cell cluster in the rows and the bulk reference transcriptome in the columns. (F). Top 20 positive and negative gene loadings of PC1 for T-bet + cDC2 clusters after cell-cycle correction (left panel). Scatterplot of PC1 and PC2 for T-bet + cDC2 clusters after cell-cycle correction (right panel).

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Expressing, FACS, Gene Expression

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet: Environmental Cues Drive Distinct DC2 Differentiation Pathways within the Spleen, Related to Figure 5 (A). Gating strategy for the identification of DC progenitors in the bone marrow (BM) (B). Palantir pseudo-time analysis of differentiation potential and branch probabilities from the Siglec-H + pre-DC state to T-bet + cDC2 and T-bet – cDC2 terminal states. (C). Plots showing Palantir differentiation potential (y axis) along Palantir pseudo-time (x axis) for Siglec-H + DC and T-bet + cDC2s (top) or Siglec-H + DC and T-bet – cDC2 clusters (bottom) (D). Plots showing the top two diffusion component embeddings for Siglec-H + DC and T-bet + cDC2 clusters (top) or Siglec-H + DC and Tbet – cDC2 clusters (bottom). Black arrow indicates Siglec-H + DC cluster cells adjacent to cells from the proliferative T-bet + cDC2 clusters 6 and 8. (E). Top panel: plots showing probability of each cell being within 20 nearest neighbors of randomly sampled shortest paths from the Siglec-H + DC to the indicated end points. Middle panel: plots showing the proportion of cells belonging to Siglec-H + DC, T-bet + cDC2, or T-bet – cDC2 from 20 nearest neighbors of randomly sampled shortest paths. Bottom: plots showing diffusion distance step sizes for each step along the indicated shortest paths (bottom panel). Colors illustrate cluster membership. (F). Graph showing AUC (x axis) for genes differentially expressed between Siglec-H + DC cluster (cluster 11) and all other cDC2 clusters. EMD on the y axis. Dashed lines represents μ EMD ± 3σ EMD . (G). Gating strategy for FACS-isolation of MHCII + ILC3s: Lin = CD3, CD19, CD49b, Siglec-F. (H). Heatmap reports scaled expression of 3550 differentially expressed genes (log 2 FC > 1, FDR < 0.01) between ILC3s and Rorγt fm cDC2s. Selected genes listed to the right. (I). Representative flow cytometric analysis of phenotypes of splenic progeny from Tbx21 RFP-cre CD45.2 + Ly6C − CD64 – MHCII + CD11c + Siglec-H + pre-DCs adoptively transferred into sub-lethally irradiated CD45.1 recipient mice 7 days earlier (data from one experiment with n = 3). J. Sort purified T-bet + or T-bet – cDC2 were cultured for 24hrs in the presence of LPS, CpG, TNF-α or IFN−γ. Representative overlay histogram showing the expression of RFP(T-bet) at 24hrs. Data representative of 2 (TNF-α) or 4 (all other cytokines/TLR agonists) independent experiments, n = 2-3.

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Diffusion-based Assay, Isolation, Expressing, Irradiation, Purification, Cell Culture

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet: Human DC Heterogeneity, Related to Figure 7 (A). Violin plots showing expression distribution of mouse DC subset marker genes across human peripheral blood DC and monocyte clusters identified in Villani et al. (2017) . (B). Representative flow cytometric analysis of mouse peripheral blood cDC2s showing absence of T-bet (RFP) + cDC2s. (C). Gating strategy for FACS-isolation of human spleen DCs for scRNA-seq. DCs were defined as live, LIN(CD3,CD56,CD19) − CD14 – CD11C + HLA-DR + . (D). Representative flow cytometry analysis of human spleen cDC2s gated as Lin(CD3,CD56,CD19) – CD14 – CD11c + HLA-DR + CD123 – XCR1 – CLEC4A + cells. Left panel: cell surface expression of CD1c and CLEC10A by cDC2s. Right panel: overlay of CLEC10A + and CLEC10A – cDC2s distinguished by differential expression of CLEC4A and FcεR1a. Summary bar graphs show frequency of CD1C + CLEC10A + and CD1C + CLEC10A – cDC2s as a percentage of cDC2s (n = 4 individuals). (E). t -SNE embedding of 9,315 FACS-isolated CD45 + immune cells from two melanoma tumors. Colors indicate unsupervised clustering by Phenograph (left panel) or classification based on expression of canonical markers and correlations with bulk RNA-seq data (right panel). Each dot represents an individual cell. (F). Pearson correlations between cluster centroids in (F) and bulk RNA-seq data from purified immune populations ( Jeffrey et al., 2006 , Novershtern et al., 2011 ) (G). t-SNE map of 2,122 myeloid cells identified in (F). Colors indicate patient sample (left) or unsupervised clustering by Phenograph (right panel). Each dot represents an individual cell. (H). Heatmap of normalized, log transformed and MAGIC imputed expression of top 20 differentially expressed genes, defined by the highest earth mover’s distance (EMD), per Phenograph cluster in E. The colored bar at the top of the heatmap shows assignment of cells to clusters labeled in F, right panel. (I). t-SNE map of human melanoma myeloid cells (H) colored by imputed expression of labeled genes.

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Expressing, Marker, Isolation, Flow Cytometry, Quantitative Proteomics, RNA Sequencing, Purification, Transformation Assay, Labeling

Journal: Cell

Article Title: Transcriptional Basis of Mouse and Human Dendritic Cell Heterogeneity

doi: 10.1016/j.cell.2019.09.035

Figure Lengend Snippet:

Article Snippet: Anti-mouse CD3ε (PE-Cy7) , Tonbo Biosciences , Cat#60-0031; RRID: AB_2621824 ; Clone 145-2C11.

Techniques: Recombinant, Staining, Multiplex Assay, Cell Isolation, Gene Expression, Software