Journal: Scientific reports

Article Title: Preclinical pharmacology characterization of HX009, a novel PD1 x CD47 Bi-specific antibody.

doi: 10.1038/s41598-024-79865-3

Figure Lengend Snippet: Fig. 1. HX009 structure and in vitro characterization. (A) Molecular structure of HX009: 2 × 2 symmetric BsAb molecule; (B) HX009 binding to the recombinant CD47 proteins of different species including human, cynomolgus monkey (upper) and mouse (lower), binding EC50 (half maximal effective concentration) were determined per four-parameter equation fitting curves; (C) competitive binding to CD47 single-positive (upper panel) or CD47-PD1 double positive (lower panel) cells by HX009. Labeled B6H12 (anti-CD47) was competed out by SIRPα-Fc, but not by HX009 (weakened binding) in single-positive cell assay, while it was competed out in double-positive cell assays by both SIPRα-Fc and HX009. (D) T-cell activation luciferase report assays: upper panel: HX009 had enhanced T-cell activation (cis-binding) over HX008 (4X), whereas the enhancement is diminished by an anti-SIRPα neutralizing antibody but not by CD47-Fc (soluble CD47). Lower panel: there was no enhancement of T-cell activation by HX008 and SIRPα-Fc combo treatment due to trans-binding.

Article Snippet: By coupling the 7.5 μg/mL of HX009 antibody on the chip surface, the recombinant human (Atagenix), cynomolgus monkey (Acrobiosystems), mouse (Sinobiological) and rat (Sinobiological) CD47 proteins, at serial loading concentrations from 2 to 128 nM (two-fold dilution), or the recombinant human (Atagenix), cynomolgus monkey (Sinobiological), mouse (Sinobiological) and rat (Sinobiological) PD1 proteins at serial loading concentrations from 1 to 64 nM (two-fold dilution), flowed through the chip surface, a binding of 200-s to reach the target level above 770 RU, and a 20-min dissociation time with 10 mM of Glycine pH 1.7.

Techniques: In Vitro, Binding Assay, Recombinant, Concentration Assay, Labeling, Activation Assay, Luciferase

Journal: Scientific reports

Article Title: Preclinical pharmacology characterization of HX009, a novel PD1 x CD47 Bi-specific antibody.

doi: 10.1038/s41598-024-79865-3

Figure Lengend Snippet: Fig. 3. HX009 anti-tumor pharmacology studies in preclinical cancer models. (A) Pharmacology evaluation of HX009 in a humanized MC38-huCD47 mouse colon cancer model in huPD1-HuGEMM mice; (B) Pharmacology evaluation of HX009 in a humanized MC38-huCD47 mouse colon cancer model in huPD1 × huPD-L1 × huCD47 × huSIRPα-HuGEMM model; (C) Pharmacology evaluation of HX009 in three AML-PDX models as shown in the figures. Columns from Left to right: Leukemic burden was measured as percentage of human CD45+ cells in peripheral blood; survival was displayed as in Caplan-Meier plot; Leukemic loads in different organs at the termination, as % of human CD45+. SP spleen, BM bone marrow, PB peripheral blood. Bottom: The differential expressions of CD47 per IHC and RNAseq in three AML-PDX models are shown in the bottom table. Graphs in A-D showed mean tumor volume ± standard error of the mean (SEM). Significance was calculated using one-way ANOVA with post-hoc comparisons or Welch’s t- test between treatment groups and vehicle group. ns, no significance; *p < 0.05; **p < 0.01; ***p < 0.001. (D) Correlation of OX40 mRNA levels with HX009 anti-lymphoma activity represented by TGI (tumor growth inhibition) in DLBCL-PDX trial.

Article Snippet: By coupling the 7.5 μg/mL of HX009 antibody on the chip surface, the recombinant human (Atagenix), cynomolgus monkey (Acrobiosystems), mouse (Sinobiological) and rat (Sinobiological) CD47 proteins, at serial loading concentrations from 2 to 128 nM (two-fold dilution), or the recombinant human (Atagenix), cynomolgus monkey (Sinobiological), mouse (Sinobiological) and rat (Sinobiological) PD1 proteins at serial loading concentrations from 1 to 64 nM (two-fold dilution), flowed through the chip surface, a binding of 200-s to reach the target level above 770 RU, and a 20-min dissociation time with 10 mM of Glycine pH 1.7.

Techniques: Activity Assay, Inhibition

Journal: eLife

Article Title: Sex-dependent gastrointestinal colonization resistance to MRSA is microbiota and Th17 dependent

doi: 10.7554/eLife.101606

Figure Lengend Snippet: ( A ) Nlrp3 gene counts from bulk RNA sequencing (RNA-seq) analysis from cells isolated from the colon lamina propria (LP) or colon epithelial cells (EC). ( B ) Il-1β gene counts from bulk RNA-seq analysis from cells isolated from the colon lamina propria (LP) or colon epithelial cells (EC). ( C ) MRSA colony forming units (CFU) in stool following oral inoculation of Nlrp3 −/− and Nlrp3 +/− mice bred at NYU. Nlrp3 −/− n=6, Nlrp3 +/− n=6. ( D ) Representative flow gating to confirm depletion of CD4+ T cells in colon lamina propria 3 days post injection. Data points represent mean ± SEM from at least two independent experiments. Statistical analysis: area under the curve followed by a two-tailed t-test. ns: not significant.

Article Snippet: C57BL/6J mice bred at NYU were injected intraperitoneally with either 200 μg rat anti-mouse Ly6G or rat IgG2a isotype control antibody to deplete neutrophils and 250 μg rat anti-mouse CD4 or rat IgG2a isotype control antibody to deplete CD4+ T cells (Bio-X-Cell, West Lebanon, NH, USA).

Techniques: RNA Sequencing, Isolation, Injection, Two Tailed Test

Journal: eLife

Article Title: Sex-dependent gastrointestinal colonization resistance to MRSA is microbiota and Th17 dependent

doi: 10.7554/eLife.101606

Figure Lengend Snippet: ( A ) Methicillin-resistant S. aureus (MRSA) colony forming units (CFU) in stool following oral inoculation of Rag2 −/− and Rag2 +/− mice bred at NYU. Male Rag2 −/− n=12, male Rag2 +/− n=12, female Rag2 −/− n=12, female Rag2 +/− n=11. ( B ) MRSA CFU in stool following oral inoculation of Ighm −/− and Ighm +/− mice bred at NYU. Male Ighm −/− n=6, male Ighm +/− n=6, female Ighm −/− n=8, female Ighm +/− n=5. ( C ) MRSA CFU in stool following oral inoculation of female NYU B6 mice injected intraperitoneally (IP) with 250 μg of anti-CD4 depleting antibody or anti-IgG control. Anti-IgG n=8, anti-CD4 n=10. Data points represent mean ± SEM from at least two independent experiments. Statistical analysis: area under the curve analyzed by a one-way ANOVA with Sidak’s multiple comparison test for ( A ) and ( B ) and a two-tailed t-test for ( C ). ns: not significant.

Article Snippet: C57BL/6J mice bred at NYU were injected intraperitoneally with either 200 μg rat anti-mouse Ly6G or rat IgG2a isotype control antibody to deplete neutrophils and 250 μg rat anti-mouse CD4 or rat IgG2a isotype control antibody to deplete CD4+ T cells (Bio-X-Cell, West Lebanon, NH, USA).

Techniques: Injection, Control, Comparison, Two Tailed Test

Journal: eLife

Article Title: Sex-dependent gastrointestinal colonization resistance to MRSA is microbiota and Th17 dependent

doi: 10.7554/eLife.101606

Figure Lengend Snippet: ( A ) Flow cytometry of cecal-colonic lamina propria CD4+ T cells as a percentage of CD45+ cells in male and female NYU mice treated with phosphate-buffered saline (PBS) or MRSA 2 days post inoculation (dpi). ( B ) Flow cytometry of cecal-colonic lamina propria IL17A+ CD4+ T cells as a percentage of total CD4+ T cells in male and female NYU mice treated with PBS or MRSA 2 dpi. ( C ) Flow cytometry of cecal-colonic lamina propria γδ T cells as a percentage of CD45+ cells in male and female NYU mice treated with PBS or MRSA 2 dpi. ( D ) Flow cytometry of cecal-colonic lamina propria IL17A+ γδ T cells as a percentage of total CD4+ T cells in male and female NYU mice treated with PBS or MRSA 2 dpi. ( E ) MRSA colony forming units (CFU) in stool following oral inoculation of Rorc −/− and Rorc +/− mice bred at NYU. Male Rorc +/− n = 5, male Rorc −/− n=7, female Rorc +/− n = 9, female Rorc −/− n=9. ( F ) MRSA CFU in stool following oral inoculation of female Il17ra +/- and Il17ra -/- mice bred at NYU. Il17ra +/- n = 6, Il17ra -/- n=6. ( G ) MRSA CFU in stool following oral inoculation of Tcrd +/− and Tcrd −/− mice bred at NYU. Male Tcrd +/− n = 6, male Tcrd −/− n=6, female Tcrd +/− n=8, and female Tcrd −/− n=12. ( H ) Flow cytometry of cecal-colonic lamina propria Ly6G+CD11b+ neutrophils as a percentage of CD45+ cells in male and female NYU mice treated with PBS or MRSA 2 dpi. ( I ) Quantification of mean fluorescence intensity (MFI) of surface CD11b on neutrophils by flow cytometry normalized to mock-treated controls. ( J ) MRSA CFU in stool following oral inoculation of NYU B6 mice treated with anti-Ly6G neutrophil depleting antibody or anti-IgG control. Male anti-IgG n=6, male anti-Ly6G n=8, female anti-IgG n = 12, and female anti-CD4+ n = 15. Data points represent mean ± SEM from at least two independent experiments. Statistical analysis: two-way ANOVA+Sidak’s multiple comparisons test for ( A – D ) and ( H ), area under the curve followed by a one-way ANOVA+Sidak’s multiple comparisons test for ( E ), ( G ), ( J ) or a two-tailed t-test for ( F ) and a two-tailed t-test for ( I ). ns: not significant.

Article Snippet: C57BL/6J mice bred at NYU were injected intraperitoneally with either 200 μg rat anti-mouse Ly6G or rat IgG2a isotype control antibody to deplete neutrophils and 250 μg rat anti-mouse CD4 or rat IgG2a isotype control antibody to deplete CD4+ T cells (Bio-X-Cell, West Lebanon, NH, USA).

Techniques: Flow Cytometry, Saline, Fluorescence, Control, Two Tailed Test

Journal: eLife

Article Title: Sex-dependent gastrointestinal colonization resistance to MRSA is microbiota and Th17 dependent

doi: 10.7554/eLife.101606

Figure Lengend Snippet: ( A ) Flow cytometry gating scheme for innate lymphoid cells (ILCs), CD4+ and γδ T cell populations. ( B ) Representative sample gating of IL17A+ CD4+ populations in unstimulated, mock and 2 days post inoculation (dpi) methicillin-resistant S. aureus (MRSA)-treated samples.

Article Snippet: C57BL/6J mice bred at NYU were injected intraperitoneally with either 200 μg rat anti-mouse Ly6G or rat IgG2a isotype control antibody to deplete neutrophils and 250 μg rat anti-mouse CD4 or rat IgG2a isotype control antibody to deplete CD4+ T cells (Bio-X-Cell, West Lebanon, NH, USA).

Techniques: Flow Cytometry

Journal: eLife

Article Title: Sex-dependent gastrointestinal colonization resistance to MRSA is microbiota and Th17 dependent

doi: 10.7554/eLife.101606

Figure Lengend Snippet: ( A ) Flow cytometry of small intestinal (SI) lamina propria CD4+ T cells as a percentage of CD45+ cells in male and female NYU mice treated with phosphate-buffered saline (PBS) or methicillin-resistant S. aureus (MRSA) 2 days post inoculation (dpi). ( B ) Flow cytometry of SI lamina propria IL-17A+ CD4+ T cells as a percentage of total CD4+ T cells in male and female NYU mice treated with PBS or MRSA 2 dpi. ( C ) Flow cytometry of cecal-colonic lamina propria CD127+ innate lymphoid cells (ILCs) as a percentage of CD45+ in male and female NYU mice treated with PBS or MRSA 2 dpi. ( D ) Flow cytometry of cecal-colonic lamina propria IL17A+ ILCs as a percentage of total ILCs in male and female NYU mice treated with PBS or MRSA 2 dpi. ( E ) Representative flow gating of CD45+ Ly6G+ cells isolated from the spleens of B6 NYU mice treated with anti-Ly6G neutrophil depleting antibody or anti-IgG control. ( F ) Representative flow cytometry gating plot of CD45+Ly6G+CD11b+ neutrophils isolated from the cecal-colonic tissue of B6 NYU mice treated with a PBS mock control or MRSA. Representative flow cytometry gating plot of CD11b mean fluorescent intensity (MFI) of Ly6G+CD11b+ neutrophils. Data points represent mean ± SEM from at least two independent experiments. Statistical analysis: two-way ANOVA+Sidak’s multiple comparisons test for ( A – D ). ns: not significant.

Article Snippet: C57BL/6J mice bred at NYU were injected intraperitoneally with either 200 μg rat anti-mouse Ly6G or rat IgG2a isotype control antibody to deplete neutrophils and 250 μg rat anti-mouse CD4 or rat IgG2a isotype control antibody to deplete CD4+ T cells (Bio-X-Cell, West Lebanon, NH, USA).

Techniques: Flow Cytometry, Saline, Isolation, Control

Journal: eLife

Article Title: Sex-dependent gastrointestinal colonization resistance to MRSA is microbiota and Th17 dependent

doi: 10.7554/eLife.101606

Figure Lengend Snippet: ( A ) MRSA colony forming units (CFU) in stool following oral inoculation of male or female mice that were irradiated and reconstituted with bone marrow (BM) from donor male or female mice. Female BM into female recipients (F→F) n=8, male BM into male recipients (M→M) n=7, female BM into male recipients (F→M) n=8. ( B ) MRSA CFU in stool following oral inoculation of ovariectomized female mice or sham operated littermate controls. Ovariectomized (OVX) n=10, sham n=10. ( C ) MRSA CFU in stool following oral inoculation of Esr1 +/- and Esr1 -/- female mice bred at NYU. Esr1 +/ - n = 6, Esr1 -/- n=6. ( D ) MRSA CFU in stool following oral inoculation of four core genotype mice. XX n=5, XY( -Sry ) n=5. XY n=5, XX( +Sry ) n=4. ( E ) CD4+ T cells from cecal-colonic lamina propria of XX females and XX( +Sry ) males 2 days post inoculation (dpi) inoculation with MRSA or mock control. ( F ) Percentage of IL-17A+CD4+ T cells in cecal-colonic lamina propria of XX females and XX(+ Sry ) males 2 dpi inoculation with MRSA or mock control. Data points represent mean ± SEM from at least two independent experiments. Statistical analysis: area under the curve followed by a one-way ANOVA with Sidak’s multiple comparisons for ( A , C ) and two-tailed t-test for ( B , F ) and two-way ANOVA+Sidak’s multiple comparisons test for ( D – E ). ns: not significant.

Article Snippet: C57BL/6J mice bred at NYU were injected intraperitoneally with either 200 μg rat anti-mouse Ly6G or rat IgG2a isotype control antibody to deplete neutrophils and 250 μg rat anti-mouse CD4 or rat IgG2a isotype control antibody to deplete CD4+ T cells (Bio-X-Cell, West Lebanon, NH, USA).

Techniques: Irradiation, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Type 1 lymphocytes and interferon-γ accumulate in the thalamus and restrict seizure susceptibility after traumatic brain injury

doi: 10.1101/2024.12.28.630606

Figure Lengend Snippet: A) Schematic of controlled cortical impact (CCI) model of TBI approximately 21 days post injury (dpi), depicting cortical and thalamic gliosis (astrocytes and microglia) and immune infiltrates (leukocytes) across a coronal section of the murine brain. B) Schematic depicting kinetics of non-B lymphocyte infiltration into cortex (black line) and thalamus (blue line) following TBI. C) Number of non-B lymphocytes (CD45 + CD11b - CD19 - Thy1.2 + ) in the perilesional cortex (left, black) and ipsilateral thalamus (right, blue) after TBI. Mice per timepoint: sham (0 dpi), 7 dpi, 14 dpi, 30 dpi n = 7 per timepoint; 42 dpi n = 6; 79 dpi n = 5. See for gating strategy. D) Number of CD8 + T cells (left) and CD4 + FoxP3 - T conv cells (right) in the perilesional cortex across a timecourse following TBI. Mice per timepoint: sham (0 dpi), 7 dpi, 14 dpi, 30 dpi n = 7 per timepoint; 42 dpi n = 6; 79 dpi n = 5. E) Number of CD8 + T cells (left) and CD4 + FoxP3 - T conv cells (right) in the ipsilateral thalamus across a timecourse following TBI. Mice per timepoint: sham (0 dpi), 7 dpi, 14 dpi, 30 dpi n = 7 per timepoint; 42 dpi n = 6; 79 dpi n = 5. F) Number of NK/ILC1 (NK1.1 + ), CD8 + T, CD4 + FoxP3 - T conv , and CD4 + FoxP3 + T reg cells in the perilesional cortex across a timecourse after TBI. Data represents means. Mice per timepoint: sham (0 dpi), 7 dpi, 14 dpi, 30 dpi n = 7 per timepoint; 42 dpi n = 6; 79 dpi n = 5. G) Representative histograms of Tbet, GATA-3, and RORγt expression within cortical CD4 + FoxP3 - T conv cells at 7 dpi. H) Quantification of Tbet + , GATA-3 + , and RORγt + frequencies within cortical CD4 + FoxP3 - T conv cells across timecourse following TBI. Mice per timepoint: sham (0 dpi), 7 dpi, 14 dpi, 30 dpi n = 7 per timepoint; 42 dpi n = 6; 79 dpi n = 5. One-way ANOVA with Tukey’s multiple comparisons test within each timepoint. I) Number of NK/ILC1 (NK1.1 + ), CD8 + T, CD4 + FoxP3 - T conv , and CD4 + FoxP3 + T reg cells in the ipsilateral thalamus across a timecourse after TBI. Data represents means. Mice per timepoint: sham (0 dpi), 7 dpi, 14 dpi, 30 dpi n = 7; 42 dpi n = 6; 79 dpi n = 5. J) Representative histograms of Tbet, GATA-3, and RORγt expression within thalamic CD4 + FoxP3 - T conv cells at 30 dpi. K) Quantification of Tbet + , GATA-3 + , and RORγt + frequencies within thalamic CD4 + FoxP3 - T conv cells across a timecourse following TBI. Mice per timepoint: sham (0 dpi), 7 dpi, 14 dpi, 30 dpi n = 7 per timepoint; 42 dpi n = 6; 79 dpi n = 5. One-way ANOVA with Tukey’s multiple comparisons test within each timepoint. L) Representative confocal images of thalami from sham (top) or TBI (21 dpi, bottom) mice, highlighting T cells (white circles, CD45 + CD3ε + surface reconstruction) and thalamic subregions (white dashed lines). Parvalbumin (PV, yellow) is included to highlight the reticular thalamic nucleus (nRT). Insets (red dashed lines, right) depict native CD45 (cyan), PV (yellow), and CD3ε (magenta) stains. M) Number of T cells (CD45 + CD3ε + cells) normalized per area of thalamic subregions from sham versus TBI (21 dpi) mice. Sham n = 4; TBI n = 8. Two-way ANOVA with Šidák’s multiple comparisons test. N) Percentage of total thalamic T cells (CD45 + CD3ε + cells) found within each thalamic subregion from TBI (21 dpi) mice. n = 8. One-way ANOVA with Tukey’s multiple comparisons test. Data are mean ± SD unless otherwise specified. Data points represent individual mice. Statistics: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Rat anti-mouse CD4 antibodies (250 μg, clone GK1.5, BioXCell) and the corresponding isotype control rat IgG2b antibodies (250 μg, clone LTF-2, BioXCell) were diluted in sterile PBS buffer (BioXcell) immediately prior to i.p. injection 3 hours after mice underwent TBI/sham surgeries.

Techniques: Expressing

Journal: bioRxiv

Article Title: Type 1 lymphocytes and interferon-γ accumulate in the thalamus and restrict seizure susceptibility after traumatic brain injury

doi: 10.1101/2024.12.28.630606

Figure Lengend Snippet: A) Gating strategy used to identify brain-infiltrating lymphocytes for . Representative flow plots are from cortical samples at 7 dpi. B) Number of B cells (left), NK/ILC1s (middle), and CD4 + FoxP3 + T regs (right) in the perilesional cortex across a timecourse after TBI. Mice per timepoint: sham (0 dpi), 7 dpi, 14 dpi, 30 dpi n = 7 per timepoint; 42 dpi n = 6; 79 dpi n = 5. C) Representative flow histograms depicting Tbet expression in B cells, NK/ILC1s, CD8 + T cells, and CD4 + FoxP3 - T conv cells from the cortex at 7 dpi, demonstrating productive Tbet staining in type 1 lymphocytes but not B cells. D) Number of B cells (left), NK/ILC1s (middle), and CD4 + FoxP3 + T regs (right) in the ipsilateral thalamus across a timecourse after TBI. Mice per timepoint: sham (0 dpi), 7 dpi, 14 dpi, 30 dpi n = 7; 42 dpi n = 6; 79 dpi n = 5. E) Representative flow histograms depicting Tbet expression in B cells, NK/ILC1s, CD8 + T cells, and CD4 + FoxP3 - T conv cells from the thalamus at 30 dpi, demonstrating productive Tbet staining in type 1 lymphocytes but not B cells. F) Number of non-B lymphocytes (CD45 + CD11b - CD19 - Thy1.2 + ) in the ipsilateral hippocampus after TBI. n = 7 mice per timepoint. G) Number of non-B lymphocytes (CD45 + CD11b - CD19 - Thy1.2 + ) in the perilesional cortex, ipsilateral hippocampus, and ipsilateral thalamus after TBI. Data represents means. n = 7 mice per timepoint. H) Representative confocal image (left) of the thalamus from Tbet zsGreen reporter mice highlighting Tbet + T cells (green squares, Tbet + CD3ε + surface reconstruction). Insets (right) depict merged and separated native Tbet and CD3ε signals.

Article Snippet: Rat anti-mouse CD4 antibodies (250 μg, clone GK1.5, BioXCell) and the corresponding isotype control rat IgG2b antibodies (250 μg, clone LTF-2, BioXCell) were diluted in sterile PBS buffer (BioXcell) immediately prior to i.p. injection 3 hours after mice underwent TBI/sham surgeries.

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: Type 1 lymphocytes and interferon-γ accumulate in the thalamus and restrict seizure susceptibility after traumatic brain injury

doi: 10.1101/2024.12.28.630606

Figure Lengend Snippet: A) Concatenated flow plots of thalamic CD8 + T cells at each timepoint depicting expression of CD44, CD62L, CD69, and CD103. B) Quantification of CD44, CD62L, CD69, and CD103 expression within thalamic CD8 + T cells at each timepoint displayed as frequency of CD8 + T cells at each timepoint. Mice per timepoint: 7 dpi n = 6; 14 dpi n = 7; 35 dpi n = 7. C) Concatenated flow plots of thalamic CD4 + T cells at each timepoint depicting expression of CD44, CD62L, CD69, and CD103. D) Quantification of CD44, CD62L, CD69, and CD103 expression within thalamic CD4 + T cells at each timepoint, displayed as frequency of CD4 + T cells at each timepoint. Mice per timepoint: 7 dpi n = 6; 14 dpi n = 7; 35 dpi n = 7. E) Representative flow histograms depicting expression of CD62L, CD44, CD69, CD103, KLRG1, and Tbet within cortical (black), thalamic (blue), and blood (red) CD8 + T cells at 14 dpi. F) Quantification of CD44, CD62L, CD69, and CD103 expression within cortical CD8 + T cells at each timepoint, displayed as frequency of CD8 + T cells at each timepoint. n = 7 mice per timepoint. G) Quantification of CD44, CD62L, CD69, and CD103 expression within cortical CD4 + T cells at each timepoint, displayed as frequency of CD4 + T cells at each timepoint. n = 7 mice per timepoint. Data are mean ± SD. Data points represent individual mice.

Article Snippet: Rat anti-mouse CD4 antibodies (250 μg, clone GK1.5, BioXCell) and the corresponding isotype control rat IgG2b antibodies (250 μg, clone LTF-2, BioXCell) were diluted in sterile PBS buffer (BioXcell) immediately prior to i.p. injection 3 hours after mice underwent TBI/sham surgeries.

Techniques: Expressing

Journal: bioRxiv

Article Title: Type 1 lymphocytes and interferon-γ accumulate in the thalamus and restrict seizure susceptibility after traumatic brain injury

doi: 10.1101/2024.12.28.630606

Figure Lengend Snippet: A) Quantification of IFNγ within the ipsilateral thalamus at 7, 14, and 35 dpi via Luminex on micro-dissected homogenates, normalized per mg of tissue. Mice per timepoint: 7 dpi n = 5; 14 dpi n = 5; 35 dpi n = 6. One-way ANOVA with Tukey’s multiple comparisons test. B) Quantification of IFNγ within the perilesional cortex, ipsilateral hippocampus, and ipsilateral thalamus at 7 dpi via Luminex on micro-dissected homogenates, normalized per mg of tissue. Mice per brain region: cortex n = 6; hippocampus n = 3; thalamus n = 5. One-way ANOVA with Tukey’s multiple comparisons test. C) Quantification of IFNγ within the perilesional cortex, ipsilateral hippocampus, and ipsilateral thalamus at 14 dpi via Luminex on micro-dissected homogenates, normalized per mg of tissue. Mice per brain region: cortex n = 6; hippocampus n = 4; thalamus n = 5. One-way ANOVA with Tukey’s multiple comparisons test. D) Quantification of IFNγ within the perilesional cortex, ipsilateral hippocampus, and ipsilateral thalamus at 35 dpi via Luminex on micro-dissected homogenates, normalized per mg of tissue. Mice per brain region: cortex n = 7; hippocampus n = 5; thalamus n = 6. One-way ANOVA with Tukey’s multiple comparisons test. E) Representative flow plots depicting IFNγ, IL-5/13, and IL-17A expression (top row) within thalamic CD45 hi CD11b - CD19 - cells at 41 dpi following ex vivo stimulation. IFNγ + cells are further gated into NK/ILC1s, CD4 + T cells, and CD8 + T cells (bottom row), depicting the relative abundance of these cell types within the total IFNγ + pool. F) Quantification of total IFNγ + , IL-5/13 + , and IL-17A + cells within the thalamus of sham or TBI mice at 30 dpi, depicted as cell numbers (left) and frequency of total CD45 + cells (right). Mice per condition: sham n = 4; TBI n = 5. Two-way ANOVAs with Šidák’s multiple comparisons tests. G) Quantification of total IFNγ + , IL-5/13 + , and IL-17A + cells within the thalamus of sham or TBI mice at 41 dpi, depicted as cell numbers (left) and frequency of total CD45 + cells (right). n = 5 mice per condition. Two-way ANOVAs with Šidák’s multiple comparisons tests. H) Number of IFNγ + , IL-5/13 + , and IL-17A + NK/ILC1, CD4 + T, and CD8 + T cells within the thalamus at 30 dpi. TBI n = 5 mice. I) Number of IFNγ + , IL-5/13 + , and IL-17A + NK/ILC1, CD4 + T, and CD8 + T cels within the thalamus at 41 dpi. TBI n = 5 mice. J) Frequency of NK/ILC1, CD4 + T cells, and CD8 + T cells within total IFNγ + cells in the thalamus at 30 dpi. Mice per condition: sham n = 4; TBI n = 5. One-way ANOVA with Tukey’s multiple comparisons test. TBI n = 5 mice. K) Frequency of NK/ILC1, CD4 + T cells, and CD8 + T cells within total IFNγ + cells in the thalamus at 41 dpi. TBI n = 5 mice. One-way ANOVA with Tukey’s multiple comparisons test. Data are mean ± SD. Data points represent individual mice. Statistics: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Rat anti-mouse CD4 antibodies (250 μg, clone GK1.5, BioXCell) and the corresponding isotype control rat IgG2b antibodies (250 μg, clone LTF-2, BioXCell) were diluted in sterile PBS buffer (BioXcell) immediately prior to i.p. injection 3 hours after mice underwent TBI/sham surgeries.

Techniques: Luminex, Expressing, Ex Vivo

Journal: bioRxiv

Article Title: Type 1 lymphocytes and interferon-γ accumulate in the thalamus and restrict seizure susceptibility after traumatic brain injury

doi: 10.1101/2024.12.28.630606

Figure Lengend Snippet: A) Quantification of IFNγ within the perilesional cortex, ipsilateral hippocampus, and ipsilateral thalamus at 7, 14, and 35 dpi via Luminex on micro-dissected homogenates, normalized per mg of tissue. B) Quantification of IL-13 within the perilesional cortex, ipsilateral hippocampus, and ipsilateral thalamus at 7, 14, and 35 dpi via Luminex on micro-dissected homogenates, normalized per mg of tissue. C) Quantification of IL-2 within the perilesional cortex, ipsilateral hippocampus, and ipsilateral thalamus at 7, 14, and 35 dpi via Luminex on micro-dissected homogenates, normalized per mg of tissue. D) Gating strategy used to identify brain-infiltrating lymphocytes for cytokine quantification following ex vivo stimulation (in ). Representative flow plots are from cortical samples at 41 dpi. E) Representative flow plots depicting IFNγ expression within thalamic NK/ILC1s, CD4 + T cells, and CD8 + T cells at 41 dpi. F) Frequency of IFNγ + , IL-5/13 + , and IL-17A + NK/ILC1s, CD4 + T cells, and CD8 + T cells at 30 dpi. TBI n = 5 mice. G) Frequency of IFNγ + , IL-5/13 + , and IL-17A + NK/ILC1s, CD4 + T cells, and CD8 + T cells at 41 dpi. TBI n = 5 mice. H) Total number of IFNγ + , IL-5/13 + , and IL-17A + cells within the perilesional cortex and ipsilateral thalamus at 41 dpi. n = 5 mice per brain region. Two-way ANOVA with Šidák’s multiple comparisons test. I) Frequency of total IFNγ + , IL-5/13 + , and IL-17A + cells (within the total CD45+ pool) in the perilesional cortex and ipsilateral thalamus at 41 dpi. Two-way ANOVA with Šidák’s multiple comparisons test. J) Frequency of NK/ILC1, CD4 + T cells, and CD8 + T cells within total IFNγ + cells in the perilesional cortex and ipsilateral thalamus at 41 dpi. n = 5 mice per brain region. Data are mean ± SD. Data points represent individual mice. Statistics: ns = not significant, *p < 0.05.

Article Snippet: Rat anti-mouse CD4 antibodies (250 μg, clone GK1.5, BioXCell) and the corresponding isotype control rat IgG2b antibodies (250 μg, clone LTF-2, BioXCell) were diluted in sterile PBS buffer (BioXcell) immediately prior to i.p. injection 3 hours after mice underwent TBI/sham surgeries.

Techniques: Luminex, Ex Vivo, Expressing

Journal: bioRxiv

Article Title: Type 1 lymphocytes and interferon-γ accumulate in the thalamus and restrict seizure susceptibility after traumatic brain injury

doi: 10.1101/2024.12.28.630606

Figure Lengend Snippet: A) Schematic depicting anti-CD4 antibody (or IgG2b isotype) treatment following sham or TBI surgeries and pentylenetetrazol (PTZ) dosing regimen to assess seizure susceptibility. Mice were initially treated with antibodies (250 μg/mouse i.p.) 3 hours after surgery, and then every 4 days for a total of 4 doses. Mice were given a sensitizing dose of PTZ (45 mg/kg i.p.) at 4 weeks post injury (wpi), and then given a challenge of PTZ (45 mg/kg i.p.) at 5 wpi, at which point seizure behavior was recorded (for 20 minutes) and quantified. B) Number of thalamic CD4 + T cells at 14 dpi, demonstrating efficient depletion of CD4 + T cells with anti-CD4 treatment. One-way ANOVA with Tukey’s multiple comparisons test. C) Quantification of seizure incidence at 5wpi, depicted as the percentage of each experimental group that experienced one or more generalized tonic-clonic (GTC) seizures (black bars). Numbers within the bar graph represent the number of mice in each group that experienced 0 or 1+ GTC seizures. Mice per condition: sham+IgG n = 32, TBI+IgG n = 34, TBI+anti-CD4 n = 33. Fisher’s exact tests. D) Quantification of GTC seizure duration (Racine Stage 5) per mouse. Mice per condition: sham+IgG n = 32, TBI+IgG n = 34, TBI+anti-CD4 n = 33. D Kruskal-Wallis test with Dunn’s multiple comparisons test. E) Unsupervised clustering (UMAP plot) of single cell RNA sequencing (scRNAseq) data from CD11b + microglia/myeloid cells pooled from sham+IgG, TBI+IgG, and TBI+anti-CD4 conditions at 14 dpi and colored by cluster (resolution = 0.5). Cells were categorized into homeostatic microglia (H1, H2), disease-associated microglia (DAM1, DAM2), interferon-responsive microglia (IFN), microglia enriched for neuronal transcripts (neuro), border-associated macrophages (BAM), and proliferative microglia (Cyc). F) UMAP plot of scRNAseq data from CD11b + microglia/myeloid cells, showing 2,500 cells per condition and colored by cluster as in (E) . G) Expression of cluster defining genes across clusters in scRNAseq data from (E,F) . Data represents RNA counts and is depicted as percent of cluster expressing each gene (circle size) and relative averaged expression (heatmap color). H) Quantification of the frequency of each cluster (out of total myeloid cells) within each experimental condition. Clusters are normalized such that equal contribution from each sample would be shown by 3 bars at 33% (black line). I) IFNγ ‘signature’ module score per cell from DAM1, DAM2, and IFN clusters split by experimental condition. IFNγ module score is based on differentially upregulated genes from microglia treated in vivo with IFNγ (see ). Kruskal-Wallis test with Dunn’s multiple comparisons test within each cluster. J) Representative flow plots depicting CD11c and CLEC7A expression within thalamic microglia (CD45 lo CD11b + CD64 + CX3CR1 + ) at 14 dpi. See gating strategy in . K) Frequency of CD11c + CLEC7A + microglia (DAMs) within total thalamic microglia at 14 dpi. One-way ANOVA with Tukey’s multiple comparisons test. L) Median fluorescence intensity (MdFI) of CLEC7A within thalamic microglia at 14 dpi. One-way ANOVA with Tukey’s multiple comparisons test. M) Frequency of CD11c + CD9 + microglia (DAMs) within total thalamic microglia at 14 dpi. One-way ANOVA with Tukey’s multiple comparisons test. N) Representative flow plots depicting Thy1.2 + Tbet + type 1 lymphocytes in the thalamus at 14 dpi. O) Number of thalamic Thy1.2 + Tbet + type 1 lymphocytes at 14 dpi. One-way ANOVA with Tukey’s multiple comparisons test. P) Number of thalamic NK/ILC1 (NK1.1 + ) cells at 14 dpi. One-way ANOVA with Tukey’s multiple comparisons test. Q) Number of thalamic CD8 + T cells at 14 dpi. One-way ANOVA with Tukey’s multiple comparisons test. For (B, K-M, and O-Q) , data are mean ± SD. For (D) , data are mean. Data points represent individual mice, except in (E-F, H-I) where dots represent individual cells. Statistics: ns = not significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Rat anti-mouse CD4 antibodies (250 μg, clone GK1.5, BioXCell) and the corresponding isotype control rat IgG2b antibodies (250 μg, clone LTF-2, BioXCell) were diluted in sterile PBS buffer (BioXcell) immediately prior to i.p. injection 3 hours after mice underwent TBI/sham surgeries.

Techniques: RNA Sequencing Assay, Expressing, In Vivo, Fluorescence

Journal: bioRxiv

Article Title: Type 1 lymphocytes and interferon-γ accumulate in the thalamus and restrict seizure susceptibility after traumatic brain injury

doi: 10.1101/2024.12.28.630606

Figure Lengend Snippet: (A-K) Mortality and seizure behaviors measured in a PTZ challenge assay (see ) at 5 weeks post injury (wpi). See for details on Racine Stages. A) Survival curve depicting percentage of surviving mice per condition within 20 minutes of PTZ injection. B) Mortality rate of mice within 20 minutes of PTZ injection, depicted as the percentage of each experimental group that died (black bars). Numbers within the bar graph represent the number of mice in each group that died or survived. C) Seizure frequency, depicted as the percentage of each experimental group that experienced 1 (black bars) or 2+ (white bars) seizures. D) Time spent in Racine Stage 1. E) Time spent in Racine Stage 2. F) Time spent in Racine Stage 3. G) Time spent in Racine Stage 4. H) Time spent in Racine Stage 6. I) Time spent in Racine Stage 7. J) Duration spent in each Racine seizure stage. K) Latency to each Racine seizure stage. For (D-I) , data are means. For (J-K) , data are means ± SEM. Data points represent individual mice. Mice per condition (pooled from 2 independent cohorts): sham+IgG n = 32, TBI+IgG n = 34, TBI+anti-CD4 n = 33. Statistics: For (B-C) , Fisher’s exact test. For (D-I) , Kruskal-Wallis test with Dunn’s multiple comparisons test. ns = not significant, *p < 0.05.

Article Snippet: Rat anti-mouse CD4 antibodies (250 μg, clone GK1.5, BioXCell) and the corresponding isotype control rat IgG2b antibodies (250 μg, clone LTF-2, BioXCell) were diluted in sterile PBS buffer (BioXcell) immediately prior to i.p. injection 3 hours after mice underwent TBI/sham surgeries.

Techniques: Injection

Journal: bioRxiv

Article Title: Type 1 lymphocytes and interferon-γ accumulate in the thalamus and restrict seizure susceptibility after traumatic brain injury

doi: 10.1101/2024.12.28.630606

Figure Lengend Snippet: A) Gating strategy used to identify microglia across brain regions for subsequent DAM marker expression analysis. Representative flow plots are from the cortex. B) Representative flow plots from the ipsilateral thalami showing CD4 + cell depletion and NK1.1 + cell expansion with anti-CD4 treatment. C) Representative flow plots from the ipsilateral thalami showing CD4 + cell depletion and CD8α + cell expansion with anti-CD4 treatment. D) Representative flow plots depicting CD11c and CLEC7A expression within perilesional cortical microglia (CD45 lo CD11b + CD64 + CX3CR1 + ). E) Frequency of CD11c + CLEC7A + microglia within total cortical microglia. F) CD4 + T cell numbers in the perilesional cortex. G) NK/ILC1 (NK1.1 + ) cell numbers in the perilesional cortex. H) CD8 + T cell numbers in the perilesional cortex. I) Concentration of CD4 + T cells in the blood. J) Concentration of NK/ILC1 (NK1.1 + ) cells in the blood. K) Concentration of CD8 + T cells in the blood. L) Concentration of myeloid cells (CD11b + ) in the blood. M) Concentration of B cell (CD19 + ) in the blood. N) Concentration of γδ T cell (CD3 + TCRγδ + ) in the blood. Data are mean ± SD. Data points represent individual mice. Statistics: One-way ANOVAs with Tukey’s multiple comparisons test. ns = non-significant, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Rat anti-mouse CD4 antibodies (250 μg, clone GK1.5, BioXCell) and the corresponding isotype control rat IgG2b antibodies (250 μg, clone LTF-2, BioXCell) were diluted in sterile PBS buffer (BioXcell) immediately prior to i.p. injection 3 hours after mice underwent TBI/sham surgeries.

Techniques: Marker, Expressing, Concentration Assay

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: A, Histograms depicting cell surface expression of CD24 and CD47 by flow cytometry on mouse cancer cell lines. B, Correlation of CD24 and CD47 surface expression of cell lines shown in A by geometric MFI. Data shown as mean ± SD of 3 technical replicates. Simple linear regression was performed to assess correlation. C, Representative plots showing quantification of CD45+ phagocytic primary mouse macrophages co-cultured with CFSE+ KPCA.C. Co-cultures were exposed to vehicle control (PBS) or 10 ug/ml of monoclonal antibodies against mouse CD47, CD24, or the combination for 2 hours. Phagocytosis is represented as CD45+ macrophages that had engulfed CFSE+ KPCA.C cells as a percentage of the total macrophage population. D, Quantification of phagocytosis for cell lines in A . Cell lines are organized based on expression levels of each surface marker. Data represent mean ± SD of 3 technical replicates. E, Correlation of cell surface expression levels of CD47 and CD24 compared to phagocytosis upon treatment with the corresponding antibodies for each cell line. Data points depict mean ± SD from 3 replicates for each experiment. Correlation was assessed by simple linear regression. F, Representative microscopy images of GFP+ KPCA.C cells when co-cultured with primary mouse macrophages upon treatment with vehicle control (PBS), 10 ug/mL anti-CD47, 10 ug/mL anti-CD24, or the combination for 6.5 days. Top row depicts raw images of GFP+ fluorescence. Bottom row depicts purple GFP+ mask for above images used for quantification of cancer cell growth. Scale bar, 800 µm. G, Quantification of fluorescent well area from co-culture experiments for multiple cell lines after 6.5 days, organized by surface expression of CD24. Cancer cells were quantified by either green (KPCA.C, 3LL ΔNRAS, MC38) or red (238N1) fluorescent area based on their fluorophore expression. Data and means shown from one (3LL ΔNRAS, MC38) or two (238N1, KPCA.C) independent experiments with 3 technical replicates per experiment. D,G, statistical significance ns, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 determined by two-way ANOVA with Holm-Sidak multiple comparison test.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: Expressing, Flow Cytometry, Cell Culture, Control, Marker, Microscopy, Fluorescence, Co-Culture Assay, Comparison

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: A, Representative histograms demonstrating cell surface expression of CD47 and CD24 on knockouts of KPCA.C and knockdowns of 238N1 by flow cytometry. B, Representative gating of phagocytic APC CD45+ mouse macrophages when co-cultured with the indicated CFSE+ KPCA.C knockouts treated with vehicle control (PBS) for 2 hours. Phagocytic macrophages are calculated as CD45+ cells that have engulfed CFSE+ cancer cells after 2 hours as a percent of all macrophages. C,D, Quantification of phagocytosis as a percentage of the maximum phagocytic response of macrophages using KPCA.C knockout cells ( C ) or 238N1 knockdown cells ( D ) treated with vehicle control (PBS), anti-mouse CD47 antibody, anti-mouse CD24 antibody, or the combination. Data represents mean ± SD of 3 technical replicates. E,F, Quantification of fluorescent well area as a measure of GFP+ KPCA.C knockout cells ( E ) or mCherry+ 238N1 knockdown cells ( F ) growth after co-culture with primary mouse macrophages and the indicated antibodies on day 6.5. Data represents mean ± SD from two independent experiments of 3 technical replicates each. G,H, Quantification of phagocytosis using CFSE+ MC38 ( G ) or 3LL ΔNRAS ( H ) cancer cells that overexpress CD24 after co-culture with primary mouse macrophages and the indicated antibodies. Data represent mean ± SD from 3 individual experiments each containing 3 technical replicates. I, Quantification of phagocytosis using StayGold+ KPCA.C cancer cells treated with vehicle control (PBS), or anti-mouse CD24 antibody, in the absence or presence of FcR blocking reagents (Fc1, anti-mouse Truestain clone 93; Fc2, anti-mouse CD16/CD32 clone 2.4G2). Data represents mean ± SD of 3 technical replicates. ( C-H ) ns, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA with Holm-Sidak multiple comparison test.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: Expressing, Flow Cytometry, Cell Culture, Control, Knock-Out, Knockdown, Co-Culture Assay, Blocking Assay, Comparison

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: Results of scRNA-seq of sorted CD45+ immune cells from experiments using CD24 or CD47 knockout tumors. ( A,C,E ) Comparison of CD47- tumors (KPCA.C CD47 knockout, 238N1 CD47 knockout) to wild-type tumors (KPCA.C control, 238N1 control). A, Relative frequencies of immune cells from CD47- versus wild-type tumors. C, UMAP showing identified cell clusters. E, Gene set enrichment analysis showing Normalized Enrichment Scores of top Hallmark pathways. ( B,D,F ) Comparison of CD24- tumors (KPCA.C CD24 knockout, 238N1 CD24 knockout) to wild-type tumors (KPCA.C control, 238N1 control). B, Relative frequencies of immune cells from CD24- versus wild-type tumors. D, UMAP showing identified cell clusters. F, Gene set enrichment analysis showing Normalized Enrichment Scores of top Hallmark pathways.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: Knock-Out, Comparison, Control

Journal: bioRxiv

Article Title: CD47 predominates over CD24 as a macrophage immune checkpoint in cancer

doi: 10.1101/2024.11.25.625185

Figure Lengend Snippet: A, Diagram showing process for high-throughput development and functional evaluation of bispecific antibodies targeting macrophage immune checkpoints. Antibody sequences were transformed into scFvs and cloned into a knob-into-hole format using a human IgG1 Fc. Constructs targeting macrophage immune checkpoints (CD47, CD24, SIRPa, PD-1) were cloned into knob formats and crossed with tumor-binding constructs in a hole format. Bispecific antibodies (n = 77) were expressed in Expi293F cells and used for downstream biochemical and functional analysis. B, Growth of StayGold+ DLD-1 cells in co-culture with human macrophages and each bispecific antibody. Each curve represents the mean for an individual bispecific antibody from 4 replicates. Black curve with hashed lines represents mean and 95% CI of control wells . C, Anti-tumor efficacy of bispecific antibodies at approximately t = 6.5 days as evaluated by macrophage checkpoint category. *p<0.05, ****p<0.0001 by one-way ANOVA with Dunnett’s multiple comparisons test. D-F, Growth curves for each of the WTa2d1 constructs ( D ), CD24-3 constructs ( E ), or CV1 constructs ( F ). G, Representative whole-well imaging of co-cultures treated with different bispecific antibodies at approximately t = 6.5 day. Green signal depicts growth of StayGold+ DLD-1 cells. Rows contain different macrophage checkpoint arms, while columns contain different tumor-binding arms. H, Scatter plot showing binding of each bispecific antibody to human neutrophils versus red blood cells. I, Representative histograms showing binding of the indicated bispecific antibodies to human neutrophils and red blood cells.

Article Snippet: Antibodies used for experiments included: InVivoMAb anti-mouse/human/rat CD47 (IAP) clone MIAP410 (BioXCell BE0283), InVivoMAb anti-mouse CD24 clone M1/69 (BioXCell BE0360), InVivoMAb anti-human CD47 clone B6.H12 (BioXCell BE0019-1), anti-human CD24 clone ML5 (Biolegend 311102), anti-human CD24 clone SN3 (GeneTex GTX74945), cetuximab (Selleckchem A2000).

Techniques: High Throughput Screening Assay, Functional Assay, Transformation Assay, Clone Assay, Construct, Binding Assay, Co-Culture Assay, Control, Imaging

Journal: Science Advances

Article Title: Antibody nanoparticle conjugate–based targeted immunotherapy for non–small cell lung cancer

doi: 10.1126/sciadv.adi2046

Figure Lengend Snippet: ( A ) Median expression of PDL1 gene in different organs in normal and cancer tissue obtained from the TCGA dataset using Gene Expression Profiling Interactive Analysis (GEPIA) . The bar plot shows the lower PDL1 expression in LUAD and LUSC compared with normal tissue. ( B ) Anti-PDL1 (red) and DAPI (blue) staining of lung tissue micro-array (TMA) from patients with NSCLC. Each specimen of cancer and adjacent normal tissue was collected from matched patients with NSCLC. The image shows high expression of PDL1 in normal tissue compared with cancer tissue. However, the density of PDL1-high cells is equal in cancer and normal tissue because of tight cellular packing in the cancer tissue (data in the Supplementary Materials). ( C ) Median expression of CD47 gene in different organs in normal and cancer tissue according to the TCGA dataset obtained using GEPIA. The bar plot shows lower CD47 expression in LUAD and LUSC compared with normal tissue.

Article Snippet: Subsequently the reaction mixture was supplemented with either the anti-mouse/human/rat CD47 antibody (BioXCell In Vivo Mab, BE0283) or anti-mouse PDL1 (B7-H1) (BioXCell In Vivo Mab, BE0101) or a combination of both.

Techniques: Expressing, Gene Expression, Staining, Microarray

Journal: Science Advances

Article Title: Antibody nanoparticle conjugate–based targeted immunotherapy for non–small cell lung cancer

doi: 10.1126/sciadv.adi2046

Figure Lengend Snippet: ( A ) Immunostaining of lung TMA by anti-CD47 (green), anti-PDL1 (red) antibody, and DAPI (blue). The image shows most of the tumors expressing high CD47 and a limited number of tumors having higher PDL1 expression. Each tissue sample was collected from a different patient having either LUAD or LUSC. The magnified image and the analysis are described in the Supplementary Materials. ( B ) Percentage of cancer cells having PDL1-high and CD47-high in patients with LUAD. Each line represents individual tissue corresponding to each tumor specimen. The data show a poor expression of PDL1 and higher CD47 in all patients with LUAD. An example of the quantification can be found in Supplementary Materials. ( C ) Percentage of cancer cells having PDL1-high and CD47-high in patients with LUSC. The data show the expression of either PDL1 or CD47 in all patients with LUSC. No patient with high CD47 and high PDL1 has been observed. ( D ) Percentage of patients expressing either PDL1-high or CD47-high in more than 5% of cancer cells compared across different stages of LUAD and LUSC. High CD47 expression has been observed in all stages of patients with adenocarcinoma, whereas an almost equal distribution of CD47-high and PDL1-high cell populations has been observed in squamous cell carcinoma.

Article Snippet: Subsequently the reaction mixture was supplemented with either the anti-mouse/human/rat CD47 antibody (BioXCell In Vivo Mab, BE0283) or anti-mouse PDL1 (B7-H1) (BioXCell In Vivo Mab, BE0101) or a combination of both.

Techniques: Immunostaining, Expressing

Journal: Science Advances

Article Title: Antibody nanoparticle conjugate–based targeted immunotherapy for non–small cell lung cancer

doi: 10.1126/sciadv.adi2046

Figure Lengend Snippet: ( A ) Schematic representation of formulation of ADNs. A single or mixture of antibodies can be used for monospecific or bispecific ADNs. ( B ) The bar graph shows the diameter of the nanoparticles in each stage of the synthesis. Data shown as means ± SEM ( n = 3). ( C ) Change in surface charge in terms of zeta potential of the anti–CD47-PDL1-ADN during synthesis. Data presented as means ± SEM ( n = 3, one-way ANOVA followed by Tukey’s multiple comparison test). ( D ) The stability of the nanoparticle in PBS (pH = 7.2) for 45 days showed no notable change in size and zeta potential. ( E ) Schematic representation of ELISA for detecting the presence of anti-PDL1 and anti-CD47 antibodies on ADNs. ( F ) The presence of the antibody and antigen binding by nanoparticles was measured by ELISA. The data show a concentration-dependent increase in optical density, signifying the presence of both CD47 and PDL1 antibodies in the nanoparticle surface. ( G ) Representative confocal microscopy image showing the internalization of anti–CD47(FITC)-PDL1(APC)-ADN in A549 cell (scale bar, 10 μm). The image shows colocalization of the green and red fluorescence, signifying the presence of both CD47(FITC) and PDL1(APC) antibodies on the nanoparticle. ( H ) Binding of the anti–CD47-PDL1-ADN to CD47 (blue) and PDL1 (black) antigen under competitive assay conditions. ( I ) Dose-dependent association and dissociation between anti–CD47-PDL1-ADN and CD47+PDL1 antigen measured by BLI. The association was monitored for 600 s, followed by dissociation for 600 s. ( J ) Comparison between the bindings of anti–CD47-PDL1-ADNs with dual antigen CD47+PDL1 (1:1) versus single antigen, either CD47 or PDL1.

Article Snippet: Subsequently the reaction mixture was supplemented with either the anti-mouse/human/rat CD47 antibody (BioXCell In Vivo Mab, BE0283) or anti-mouse PDL1 (B7-H1) (BioXCell In Vivo Mab, BE0101) or a combination of both.

Techniques: Formulation, Zeta Potential Analyzer, Comparison, Enzyme-linked Immunosorbent Assay, Binding Assay, Concentration Assay, Confocal Microscopy, Fluorescence

Journal: Science Advances

Article Title: Antibody nanoparticle conjugate–based targeted immunotherapy for non–small cell lung cancer

doi: 10.1126/sciadv.adi2046

Figure Lengend Snippet: ( A ) Representative fluorescence microscopy image showing the internalization of the anti–CD47-PDL1-ADN (green fluorescence) in A459 cells. The image shows negligible internalization of the DNs with antibody-targeting (scale bars, 10 μm). ( B ) The bar plots show the MFI of fluorescent ADNs in A549 cells. The internalization of anti–CD47-ADN is significantly higher than that of anti–PDL1-ADN or IgG-ADN. Data show means ± SEM ( n = 3, two-way ANOVA followed by Tukey’s multiple comparison test). ( C ) Comparison of time-dependent cellular internalization of monospecific and bispecific ADNs evaluated by flow cytometry. The anti–CD47-ADN have shown higher cellular internalization than anti–PDL1-ADN because of higher cell surface CD47 antigen than PDL1 on A549 cells. Data show means ± SEM ( n = 3, two-way ANOVA followed by Tukey’s multiple comparison tests). ( D ) Cytotoxicity of the untargeted (DNs) and monospecific and bispecific ADNs in LLC cells. ( E ) The bar plot represents the IC 50 values from each ADN in the case of A549, LLC, and KLN-205 cells. The variation of the IC 50 values was observed based on the expression of CD47 and PDL1 on the cancer cells. ( F ) Representative confocal microscopy image of phagocytosis of LLC cells by mouse macrophage (RAW 264.7) in the presence of anti–CD47-PDL1-ADN (scale bar, 10 μm). ( G ) Comparison of the phagocytosis events between cancer cells and macrophages in the presence and absence of anti–CD47-PDL1-ADN. Data show means ± SEM (unpaired t test, two-tailed). ( H ) Schematic for experimental validation of immune checkpoint blocking by anti–CD47-PDL1-ADN in lung cancer cells. ( I ) The data illustrate the dose-dependent blocking of cell surface CD47 antigen in lung cancer cells, resulting in the unavailability of the free antigen for anti-CD47(FITC) mAb. ( J ) Comparison of CD47 antigen blocking by free drug, DNs, and anti–CD47-PDL1-ADN.

Article Snippet: Subsequently the reaction mixture was supplemented with either the anti-mouse/human/rat CD47 antibody (BioXCell In Vivo Mab, BE0283) or anti-mouse PDL1 (B7-H1) (BioXCell In Vivo Mab, BE0101) or a combination of both.

Techniques: Fluorescence, Microscopy, Comparison, Flow Cytometry, Expressing, Confocal Microscopy, Two Tailed Test, Biomarker Discovery, Blocking Assay

Journal: Science Advances

Article Title: Antibody nanoparticle conjugate–based targeted immunotherapy for non–small cell lung cancer

doi: 10.1126/sciadv.adi2046

Figure Lengend Snippet: ( A ) Schematic representation of the experimental design for the in vivo antitumor efficacy study of ADNs in a syngeneic LLC tumor model. ( B ) Tumor growth curves show the change in tumor volume of each treatment group. The representative images of the tumor are shown. The tumor broke into small pieces because of its friable nature. Data show means ± SEM ( n = 5, two-way ANOVA following Tukey’s multiple comparison test). ( C ) Kaplan-Meier plots show survival response. ( D ) Graph shows normalized RBC levels in each group of mice. Data show means ± SEM ( n = 3, two-way ANOVA following Tukey’s multiple comparison test). ( E ) Representative scatter plot showing intertumoral T cells in KLN-205 tumor. ( F ) The bar graph shows a significant increase in intratumoral cytotoxic T cells in mice treated with anti–CD47-PDL1-ADN. The data show means ± SEM ( n = 3, unpaired t test, two-tailed). ( G ) Immunostaining images of a tumor tissue section from control and anti–CD47-PDL1-ADN-treated groups. The color code of each marker is mentioned on the right side of the images. The images were captured using a TissueFAXS slide scanner.

Article Snippet: Subsequently the reaction mixture was supplemented with either the anti-mouse/human/rat CD47 antibody (BioXCell In Vivo Mab, BE0283) or anti-mouse PDL1 (B7-H1) (BioXCell In Vivo Mab, BE0101) or a combination of both.

Techniques: In Vivo, Comparison, Two Tailed Test, Immunostaining, Control, Marker