Journal: The Journal of Immunology Author Choice

Article Title: A Real-Time Image-Based Efferocytosis Assay for the Discovery of Functionally Inhibitory Anti-MerTK Antibodies

doi: 10.4049/jimmunol.2200597

Figure Lengend Snippet: Efferocytosis assay using cynomolgus monkey M2 macrophages. (A) In vitro differentiated cynomolgus monkey M2 macrophages (brightfield, original magnification ×10) showed phenotypes similar to those of their human counterparts. (B) A representative kinetic curve (purple) of efferocytosis mediated by monkey M2 macrophages with an image (inlet, original magnification ×10) taken at 4.5 h; assay background (pHrodo-labeled apoptotic Jurkat cells only) is shown in white circles. (C) One anti-MerTK Ab showed comparable inhibitory potencies in monkey (purple circles) versus human (blue circles) M2-mediated efferocytosis; isotype control Ab shown in gray (circles and squares represent results in monkey and human M2-mediated efferocytosis, respectively). Efferocytosis activity curves showed representative data (mean ± SD; n = 4) of three independent experiments.

Article Snippet: Cynomolgus monkey bone marrow cells (IQB-MnBM1-5) were obtained from iQ Biosciences (Berkeley, CA).

Techniques: In Vitro, Labeling, Control, Activity Assay

Journal: eLife

Article Title: Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A) cause a primary immunodeficiency disorder

doi: 10.7554/elife.72559

Figure Lengend Snippet: Figure 4. Patient T cell responses to T cell receptor stimulation. (A) Representative flow plots showing expression of IFN-γ in human PBMCs from a healthy donor and the patient. PBMCs were stimulated with anti-CD3 and anti-CD28 antibodies for 48 hr and cultured for further 4 days in the presence of IL-2 before re-stimulation with PMA plus ionomycin for 5 hr for cytokine analysis. Cells were gated for CD4+ T cells. (B) Quantitative mRNA expression analysis (± s.d.m.) of indicated cytokines from human PBMCs (cultured as mentioned above) with or without stimulation with PMA plus ionomycin for 5 hr. (C) Levels of IL-2 and TNF in human PBMCs from culture supernatants of cells stimulated as described above (B) were determined by ELISA. (D) Representative traces showing averaged (± SEM) SOCE responses from healthy control and patient PBMCs (cultured as indicated in A), after transient stimulation with anti-CD3 antibody cross-linking, or ionomycin (0.5 µM) in the presence of external solution containing 2 mM Ca2+ (left) as indicated. Bar graphs show baseline subtracted ratio values for anti-CD3 antibody cross-linking or ionomycin (average± SEM) from six independent experiments (right). (E) Representative traces showing averaged (± SEM) SOCE responses from healthy control and patient PBMCs (cultured as indicated in A), after store-depletion with thapsigargin (1 µM) stimulation in Ca2+-free Ringer’s solution. SOCE was measured by addition of 2 mM Ca2+-containing Ringer’s solution as indicated (left). Bar graph shows baseline subtracted ratio values at the peak of SOCE (average± SEM) from four independent experiments (right). (F) Phosphorylated JNK levels in CD4+ T cells from healthy control and patient PBMCs (cultured as indicated in A), stimulated with anti-CD3 antibody for indicated times. Bar graphs show average± SEM from three independent experiments. ** p < 0.005, *** p < 0.0001.

Article Snippet: PBMCs from healthy control and patient were activated for 48 hr on a plate coated with 10 μg/ml of anti- CD3 antibody (OKT3, Bio X Cell) and cultured in T cell media (DMEM containing 20% fetal bovine serum and 1% Pen- Strep) supplemented with 5 μg/ml of anti- CD28 antibody (Bio X cell), and 20 U/ml IL- 2 (Peprotech) for ThN differentiation.

Techniques: Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Control

Journal: eLife

Article Title: Biallelic mutations in calcium release activated channel regulator 2A (CRACR2A) cause a primary immunodeficiency disorder

doi: 10.7554/elife.72559

Figure Lengend Snippet: Figure 5. Cytokine expression profiles in T cells expressing individual allelic mutations in CRACR2A. (A) Representative flow plots showing expression of IL-2 in control or CRACR2A KO Jurkat T cells stably expressing FLAG-tagged WT CRACR2A, CRACR2AE278D (E278D), or CRACR2AR144G/E300* (DM) after stimulation with PMA plus ionomycin for 16 hr (left). Bar graph shows means ± s.e.m. of pooled technical replicates from two independent experiments (right). (B) Representative flow plots showing expression of IFN-γ in primary human CD4+ T cells transduced with lentiviruses encoding CRACR2A- targeting sgRNA and those encoding cDNAs of WT or indicated mutants of CRACR2A after stimulation with anti-CD3 and anti-CD28 antibodies for 5 hr (left). Bar graph (right) shows means ± s.e.m. of pooled technical replicates from three independent experiments. (C) Representative flow plots showing expression of IFN-γ in primary human CD4+ T cells purified from healthy donors and transduced with lentiviruses encoding cDNAs for WT or indicated mutant of CRACR2A after stimulation with anti-CD3 and anti-CD28 antibodies for 5 hr (left). Bar graph (right) shows means ± s.e.m. of pooled technical replicates from three independent experiments. * p < 0.05, *** p < 0.0001.

Article Snippet: PBMCs from healthy control and patient were activated for 48 hr on a plate coated with 10 μg/ml of anti- CD3 antibody (OKT3, Bio X Cell) and cultured in T cell media (DMEM containing 20% fetal bovine serum and 1% Pen- Strep) supplemented with 5 μg/ml of anti- CD28 antibody (Bio X cell), and 20 U/ml IL- 2 (Peprotech) for ThN differentiation.

Techniques: Expressing, Control, Stable Transfection, Transduction, Purification, Mutagenesis

Journal: Cell

Article Title: Therapeutic potential of co-signaling receptor modulation in hepatitis B

doi: 10.1016/j.cell.2024.05.038

Figure Lengend Snippet: Validation of co-signaling receptor modulation in independent mouse models, related to Figure 2 (A) Experimental setup. 10 6 naive Cor93 T (Cor93 T N ) cells were adoptively transferred into HBV-Tg mice (lineage MUP-core). 24 h later, indicated groups of mice were injected intraperitoneally with PBS or 100 μg of monoclonal antibodies (mAbs) blocking PD-1, LAG-3, or CTLA-4. Livers were collected and analyzed at day 5. (B) Total numbers of intrahepatic leukocytes (IHLs) isolated from the indicated mice. (C) Total numbers of Cor93 T cells in the livers of the indicated mice. (D) Representative density plots of IFN-γ expression among Cor93 T cells in the liver of the indicated mice. Numbers represent the percentage of cells within the indicated gates. (E) Total number of IFN-γ-producing Cor93 T cells in the livers of the indicated mice upon ex vivo cognate peptide stimulation. n = 3–4; one-way Brown-Forsythe and Welch ANOVA test with Dunnett correction. Each group was compared with PBS-injected controls. (F) Amount of serum alanine transaminases (sALTs) in the serum of the indicated groups of mice at the indicated time points. (G) Experimental setup. HBV replication-competent transgenic mice (lineage 1.3.32) were injected intraperitoneally with PBS or with 100 μg of agonist mAbs activating OX40 or 4-1BB. Livers were collected and analyzed at day 4. (H) Total numbers of IHL isolated from the indicated mice. (I) Representative micrographs of liver sections from the indicated groups of mice. The upper panels show hematoxylin-eosin (H&E) staining, the middle panels show immunohistochemical staining for cleaved caspase 3 (ΔCas3, brown), and the lower panels show immunohistochemical staining for HBcAg (brown). Scale bar represents 100 μm. (J) HBV DNA quantification by southern blot analysis of liver lysates from the indicated mice. Bands corresponding to the expected size of the integrated transgene (Tg), relaxed circular (RC), double-stranded (DS) linear, and single-stranded (SS) HBV DNAs are indicated. (K) Amount of sALT in the serum of the indicated groups of mice at the indicated time points. (L) Experimental setup. 10 6 Cor93 T N cells were adoptively transferred into HBV-Tg mice (lineage MUP-core). 24 h later, selected groups of mice were injected intraperitoneally with PBS or 100 μg of mAbs activating ICOS, OX40, or 4-1BB. Livers were collected and analyzed at day 5. (M) Total numbers of IHL isolated from the indicated mice. (N) Numbers of Cor93 T cells isolated from the liver of the indicated mice. (O) Representative density plots of IFN-γ expression among Cor93 T cells in the liver of the indicated mice. (P) Total number of IFN-γ-producing Cor93 T cells in the livers of the indicated mice upon ex vivo cognate peptide stimulation. n = 3–4; one-way Brown-Forsythe and Welch ANOVA test with Dunnett correction for multiple comparisons. Each group was compared with control. (Q) Amount of sALT in the serum of the indicated group of mice at the indicated time points. n = 3–4; two-way ANOVA test with Dunnett correction for multiple comparisons. Each group was compared with control group (simple effect within row). (R) Experimental setup. 10 6 naive Env28 CD8 + TCR transgenic cells (Env28 T N ) were adoptively transferred into HBV replication-competent transgenic mice (lineage 1.3.32; background C57BL/6 × BALB/c H-2 bxd hybrids). 24 h later, selected groups of mice were injected intraperitoneally with PBS or 100 μg of mAbs activating OX40 or 4-1BB. Livers were collected and analyzed at day 5. (S) Numbers of IHL isolated from the indicated mice. (T) Numbers of Env28 T cells isolated from the liver of the indicated mice. (U) Representative density plots of IFN-γ expression among Env28 T cells in the liver of the indicated mice. (V) Percentages of IFN-γ-producing Env28 T cells in the livers of the indicated mice upon ex vivo cognate peptide stimulation. n = 3–4; one-way Brown-Forsythe and Welch ANOVA test with Dunnett correction. Each group was compared with PBS-injected controls. (W) Amount of sALT in the serum of the indicated groups of mice at the indicated time points. n = 3–4; two-way ANOVA test with Dunnett correction for multiple comparisons. Each group was compared with PBS-injected controls (simple effect within row). (X) Representative micrographs of liver sections from the indicated groups of mice. The upper panels show staining for HBcAg, and the lower panels show staining for cleaved caspase 3 (ΔCas3). Scale bar represents 100 μm. (Y and Z) Representative histograms (Y) and percentages (Z) of in vitro differentiated Cor93 T effector (Cor93 T E ) or Env28 T effector (Env28 T E ) cells producing IFN-γ upon cognate in vitro peptide stimulation at the indicated concentrations. Results are representative of two independent experiments giving similar results.

Article Snippet: InVivoMAb anti-mouse/human/rat/monkey ICOS (CD278) , Bio X Cell , Cat# BE0353; RRID: AB_2894772.

Techniques: Biomarker Discovery, Injection, Bioprocessing, Blocking Assay, Isolation, Expressing, Ex Vivo, Transgenic Assay, Staining, Immunohistochemical staining, Southern Blot, Control, In Vitro

Journal: Cell

Article Title: Therapeutic potential of co-signaling receptor modulation in hepatitis B

doi: 10.1016/j.cell.2024.05.038

Figure Lengend Snippet:

Article Snippet: InVivoMAb anti-mouse/human/rat/monkey ICOS (CD278) , Bio X Cell , Cat# BE0353; RRID: AB_2894772.

Techniques: Purification, Virus, Recombinant, Staining, Saline, Fluorsave, Sequencing, DNA Labeling, Cell Isolation, Sample Prep, Reverse Transcription, Software, Microscopy