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: Communications Biology

Article Title: Tankyrase inhibition sensitizes melanoma to PD-1 immune checkpoint blockade in syngeneic mouse models

doi: 10.1038/s42003-020-0916-2

Figure Lengend Snippet: a , B16-F10 Ctnnb1 KO tumor (s.c.) end volume reduction upon anti-PD-1 (−78%) and combined anti-PD-1/G007-LK treatment (−90%) in C57BL/6 N mice treated from day 11 through day 25. Control diet ( n = 9), G007-LK diet ( n = 9), anti-PD-1 ( n = 10), and anti-PD-1/G007-LK ( n = 10). Mann-Whitney rank sum tests are indicated by † ( P < 0.05) and ‡ ( P < 0.01). For a – e Mean values are indicated by grey lines. b Tumor end volumes of B16-F10-challenged (s.c.) C57BL/6 Rag2 −/− mice treated for 12 days with control diet ( n = 11) compared to combined anti-PD-1/G007-LK treatment ( n = 10). n.s. not sig n ificant. c B16-F10 tumor (s.c.) end volume reduction upon anti-PD-1/G007-LK treatment in C57BL/6 N mice from day 10 through day 21. Control diet ( n = 11), anti-IgG1 isotype control ( n = 12), anti-IgG2 isotype control ( n = 10), anti-CD8α ( n = 10), anti-IFNγ ( n = 9), anti-PD-1/G007-LK ( n = 10), anti-PD-1/G007-LK /anti-CD8α ( n = 9), and anti-PD-1/G007-LK /anti-IFNγ ( n = 9). One-tailed t -tests are indicated by *( P ≤ 0.05) while one-tailed Mann-Whitney rank sum test is indicated by † ( P < 0.05). d Flow cytometry analysis of CD8 + and CD4 + T cells (shown as % of CD45 + T cells) from single-cell suspensions of comparable-sized tumors collected from B16-F10-challenged (s.c.) C57BL/6 N mice treated for 7–17 days with control diet ( n = 14), G007-LK diet ( n = 13), anti-PD-1 ( n = 14) or anti-PD-1/G007-LK ( n = 13). Two-tailed t -tests are indicated by *( P < 0.05) and **( P < 0.01). e CD8 + T cell counts from IHC sections from s.c. wild-type B16-F10 tumors (control diet [ n = 5], G007-LK diet [ n = 4]), and B16-F10 Ctnnb1 KO tumors (control diet [ n = 7], G007-LK diet [ n = 8]) anti-PD-1 [ n = 10] and a n ti-PD-1/G007-LK [ n = 9]). Two-tailed t -tests are indicated by *( P < 0.05) and one-tailed Mann-Whitney rank sum test is indicated by ‡ ( P < 0.01).

Article Snippet: For T cells, the following antibodies against murine targets were used: CD45 (CD45-PacBlue [30-F11], 48-0451-82, eBioscience), CD3 (CD3-Violet 605 [17A2], BioLegend), CD4 (CD4-APC-Cy7 [GK1.5], 47-0041-82, eBioscience), CD8a (CD8-PerCP [53-6.7], eBioscience), CD25 (CD25-APC [PC61.5], 17-0251-82, eBioscience) and CD44 (CD44-FITC [IM7], 11-0441-82, eBioscience).

Techniques: Control, MANN-WHITNEY, One-tailed Test, Flow Cytometry, Two Tailed Test

Journal: Stem Cell Investigation

Article Title: Creation of human hematopoietic chimeric cell (HHCC) line as a novel strategy for tolerance induction in transplantation

doi: 10.21037/sci-2022-026

Figure Lengend Snippet: Confirmation of the hematopoietic phenotype stability of the created HHCC cell line after cell fusion by expression of the hematopoietic cell surface markers. (A) Representative flow cytometry histograms of the hematopoietic and stem cell surface markers (CD34 + , CD133 + , CD117 + , CD90 + , CD4 + , CD19 + , CD14 + ) in the created HHCC at 3 and 24 hours after fusion procedure. (B) Expression of the hematopoietic cell surface markers (CD34 + , CD133 + , CD117 + , CD90 + , CD4 + , CD19 + , CD14 + ) assessed at 3 and 24 hours after cell fusion. Increased expression of CD133 + , CD4 + and CD14 + , observed at 24 hours after cell fusion. (C) Expression of the stem and progenitors’ cells surface markers including: precursor stem cells (CD133 + /CD90 + ), T-cells (CD4 + /CD8 + ), T-regulatory cells (CD4 + /CD25 + ), B-cells (CD5 + /CD19+), and dendritic cells (CD123 + /CD11c + ) evaluated at 3 hours and 7 days of cell culture after fusion. Increased expression of CD4 + /CD8 + , CD4 + /CD25 + , CD5 + /CD19 + and CD123 + /CD11c + cells was observed at 7 days after cell fusion. (D) Representative flow cytometry dotplots of T-regulatory cells surface markers (CD4 + /CD25 + ) assessed in the CD34 + naive donor cells (upper row) and in the created HHCC (lower row) at 7 days and 21 days of cell culturing after fusion procedure. HHCC, human hematopoietic chimeric cells.

Article Snippet: For HHCC staining the following anti-human monoclonal antibodies were used: CD34 (APC, BD Biosciences, NJ, USA), CD133 (APC, Miltenyi Biotech), CD117 (APC, BD Biosciences), CD90 (BV421, BioLegend, CA, USA), CD4 (APC-Cy7, BD Biosciences), CD19 (APC-Cy7, BD Biosciences), CD19 (APC, BD Biosciences) and CD14 (APC, BD Biosciences), CD133 + /CD90 + (APC, Miltenyi Biotech and BV421, BioLegend), CD4 + /CD8 + (APC-Cy7, Miltenyi Biotech and BV421, BioLegend), CD4 + /CD25 + (APC-Cy7, Miltenyi Biotech and BV421, BioLegend), CD5 + /CD19 + (BV421, BioLegend and APC-Cy7, Miltenyi Biotech), and dendritic cells CD123 + /CD11c + (APC, Miltenyi Biotech and BV421, BioLegend).

Techniques: Expressing, Flow Cytometry, Cell Culture