murine cxcl10  (Boster Bio)

Journal: Frontiers in Immunology

Article Title: PD-1 and CTLA-4 serve as major gatekeepers for effector and cytotoxic T-cell potentiation by limiting a CXCL9/10-CXCR3-IFNγ positive feedback loop

doi: 10.3389/fimmu.2024.1452212

Figure Lengend Snippet: CXCL10 and CXCL9 drive the polarization of CD4+ T cells into IFNγ high cytotoxic T cells and Th17 high CD4+ T cells while increasing their Ki-67 and IL-2 expression. (A) Spleenocytes were obtained from naïve mice (WT) or CXCL10 knockout (KO) mice (6 per group) and sorted into CD4+ cells using a MACS Easy-Sep magnetic separation kit. CD4+ cells were then cultured at a density of 10 6 cells per well, in the presence of anti-CD3 monoclonal antibody (mAb) at 5µg/ml, anti-CD28 mAb at 3µg/ml, and IL-2 at 10ng/ml. Following four days in culture, the cells were supplemented with either 100 ng per well of CXCL9 or CXCL10 for 48 hours and subsequently analyzed via flow cytometry to assess the expression of CXCR3, PD-1, IFN-γ, Ki-67, IL-2, IL-17, granzyme B, and perforin. (B) CD4+ and CD8+ T cells activated with anti-CD3 from wild-type (WT) and CXCR3 knockout (KO) mice were assessed for the production of CXCL9 and CXCL10 by ELISA. (C) CXCL10 and CXCL9 drove the polarization of CD4+ T cells towards IFNγ high Th1-like cells, Th17 effector cells, and granzyme-B high cytotoxic T cells, while also enhancing expression of Ki-67 and IL-2. Results present data of one of two independent experiments with similar observations. Significance was determined using a one-way analysis of variance (ANOVA) test using Tukey’s multiple comparisons test; *P≤ 0.05, **P≤ 0.01, ***P≤ 0.001, ****P<0.0001 were considered significant. ns, not significant.

Article Snippet: Cytokine concentrations in culture media were assessed using commercial ELISA kits: Murine IP-10 (CXCL10) Standard ABTS ELISA Development Kit, (900-K153, Peprotech), Human IP-10 (CXCL10) Standard ABTS ELISA Development Kit (900-K39, Peprotech), ELISA Kit for MOUSE CXCL9 (UCSN SEB928Mu), Human MIG (CXCL9) Standard ABTS ELISA Development Kit (900-K87, Peprotech) and Murine IFN gamma Standard ABTS ELISA Development Kit, (900-K98, Peprotech) according to manufacturer’s instructions.

Techniques: Expressing, Knock-Out, Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay

Journal: Frontiers in Immunology

Article Title: PD-1 and CTLA-4 serve as major gatekeepers for effector and cytotoxic T-cell potentiation by limiting a CXCL9/10-CXCR3-IFNγ positive feedback loop

doi: 10.3389/fimmu.2024.1452212

Figure Lengend Snippet: Blockade of PD-1 and CTLA-4 induces the CXCR3-CXCL10-IFNγ cycle in spheroids with activated CD8+ T cells coculture setup. (A) A scheme depicting the coculture protocol for MC38 spheroids with activated CD8+ T cells isolated from WT C57Bl/6 mice. MC38 spheroids were either cocultured with or without activated CD8+ T cells, supplemented with 10 µg/ml of αPD-1 or αCTLA4, and analyzed 24 hours post-treatment. (B) Flow cytometry analysis conducted on the interior compartment of the spheroid. (C) ELISA analysis of CXCL10 and IFN-γ levels following the addition of αPD-1 or αCTLA4. (D) Monitoring of spheroid structure using a light microscope. Results present data of one of three independent experiments with similar observations. Significance was determined using a one-way analysis of variance (ANOVA) test using Tukey’s multiple comparisons test; *P≤ 0.05, **P≤ 0.01, ***P≤ 0.001 were considered significant.

Article Snippet: Cytokine concentrations in culture media were assessed using commercial ELISA kits: Murine IP-10 (CXCL10) Standard ABTS ELISA Development Kit, (900-K153, Peprotech), Human IP-10 (CXCL10) Standard ABTS ELISA Development Kit (900-K39, Peprotech), ELISA Kit for MOUSE CXCL9 (UCSN SEB928Mu), Human MIG (CXCL9) Standard ABTS ELISA Development Kit (900-K87, Peprotech) and Murine IFN gamma Standard ABTS ELISA Development Kit, (900-K98, Peprotech) according to manufacturer’s instructions.

Techniques: Isolation, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Light Microscopy

Journal: Advanced Science

Article Title: Islet‐Resident Memory T Cells Orchestrate the Immunopathogenesis of Type 1 Diabetes through the FABP4‐CXCL10 Axis

doi: 10.1002/advs.202308461

Figure Lengend Snippet: T RM cells enhance the immunopathologic development of T1D through the FABP4‐CXCL10 axis. A) Representative immunoblots of CXCL10 and HSP90 in the pancreas of FABP4 +/+ NOD and FABP4 −/− NOD mice. The right panel is the quantification of the band intensity of CXCL10 relative to HSP90 ( n = 4). B) The mRNA abundance of FABP4, IFNγ, and CXCL10 in FABP4 +/+ T RM cells and FABP4 −/− T RM cells isolated from NOD mice ( n = 6). C) Representative FACS plots showing the abundance of IFNγ and CXCL10 in FABP4 +/+ T RM cells or FABP4 −/− T RM cells. The right panel is the quantification of positive cell percentage ( n = 5). D) Concentration of IFNγ and CXCL10 in the medium of FABP4 +/+ T RM cells or FABP4 −/− T RM cells stimulated with CD3 + CD28 + beads or vehicle ( n = 6). E,F) Representative images (E) and quantification (F) of displacement length (left) and average speed (right) analyzed by Imaris showing the motility of GFP‐labeled CD8 + T cells cultured in the conditioned media of FABP4 +/+ T RM or FABP4 −/− T RM cells in the presence or absence of recombinant CXCL10 protein (2 µg mL −1 ) or IgG control (2 µg mL −1 ) for 24 h (scale bar 50 µm). G) The number of residual (upper) and migrated (lower) CD8 + T cells cultured in the trans‐well plate with a conditioned medium of FABP4 +/+ T RM cells or FABP4 −/− T RM cells in the presence or absence of recombinant CXCL10 protein (2 µg mL −1 ) or IgG (2 µg mL −1 ) for 24 h ( n = 3). H,I) Representative pictures (H) and quantification (I) of displacement length (left) and average speed (right) showing the motility of GFP‐labeled CD8 + T cells cultured in the conditioned medium of FABP4 +/+ T RM or FABP4 −/− T RM cells treated with siCXCL10 (5 nmol) or siControl (siCtrl, 5 nmol) for 24 h (scale bar 50 µm). J) The number of residual (upper) and migrated (lower) CD8 + T cells cultured in the trans‐well plate with the conditioned medium from FABP4 +/+ T RM cells or FABP4 −/− T RM cells treated with siCXCL10 or siCtrl (5 nmol) for 24 h ( n = 5). Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. Unpaired Student's t test or Mann‐Whitney U test was used in (A), (B), and (C). ANOVA followed by Sidak's test was used in (D), (F), (G), (I), and (J).

Article Snippet: To assess the alarming function of FABP4 +/+ T RM and FABP4 −/− T RM cells and the involvement of CXCL10, the conditioned medium of respective cells were added to the lower wells. and supplemented with recombinant murine CXCL10 (2 g mL −1 , PeproTech) or IgG as control.

Techniques: Western Blot, Isolation, Concentration Assay, Labeling, Cell Culture, Recombinant, Control, MANN-WHITNEY

Journal: Advanced Science

Article Title: Islet‐Resident Memory T Cells Orchestrate the Immunopathogenesis of Type 1 Diabetes through the FABP4‐CXCL10 Axis

doi: 10.1002/advs.202308461

Figure Lengend Snippet: Targeting FABP4 resembles T RM cell depletion in alleviating T1D development. FABP4 +/+ NOD and FABP4 −/− NOD mice were subjected to the treatment of anti‐CD69 neutralizing antibody to deplete T RM cells. A) Schematic diagram showing the protocol of T RM cell depletion in NOD mice. Six‐week‐old female FABP4 +/+ NOD and FABP4 −/− NOD mice were injected with anti‐CD69 antibody (40 µg/mouse) or IgG every week (i.v.) for 8 weeks ( n = 5). B) The representative FACS plots show the abundance of T RM cells (CD8 + CD44 + CD69 + CD62L − ) in the mouse pancreas. The right panel is the quantification of the percentage of T RM cells ( n = 5). C) Diabetes incidence of mice ( n = 5). D) Scoring of histological grades of insulitis in mice ( n = 5). E) Representative images of immunofluorescent co‐staining for insulin‐positive β cells (green) and TUNEL‐positive apoptotic cells (red) in mouse pancreas (scale bar 20 µm). The lower panel is the quantification of the number of apoptotic cells within the islet area ( n = 5). F) The mRNA abundance of inflammatory cytokines and chemokines in mouse pancreas ( n = 5). G) The abundance of CXCL10 protein in mouse pancreatic islets ( n = 4). H) Representative immunoblots of CXCL10 and HSP90 in mouse pancreas. The lower panel is the quantification of the band intensity of CXCL10 relative to HSP90 ( n = 4). I) The abundance of total cytotoxic T cells (CD8 + CD44 + ), antigen‐experienced T cells (CD8 + CXCR3 + ), and IFN γ or Granzyme B expressing CD8 + T cells infiltrated in the pancreatic islets of the above mice ( n = 5). Data are expressed as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001. ANOVA followed by Sidak's test was used in (B), (E), (F), (G), (H), and (I). The log‐rank test was used in (C). The Pearson's chi‐square test was used in (D).

Article Snippet: To assess the alarming function of FABP4 +/+ T RM and FABP4 −/− T RM cells and the involvement of CXCL10, the conditioned medium of respective cells were added to the lower wells. and supplemented with recombinant murine CXCL10 (2 g mL −1 , PeproTech) or IgG as control.

Techniques: Injection, Staining, TUNEL Assay, Western Blot, Expressing

Journal: Advanced Science

Article Title: Islet‐Resident Memory T Cells Orchestrate the Immunopathogenesis of Type 1 Diabetes through the FABP4‐CXCL10 Axis

doi: 10.1002/advs.202308461

Figure Lengend Snippet: T RM cells orchestrate the immunopathogenesis of type 1 Diabetes through FABP4‐CXCL10 axis.

Article Snippet: To assess the alarming function of FABP4 +/+ T RM and FABP4 −/− T RM cells and the involvement of CXCL10, the conditioned medium of respective cells were added to the lower wells. and supplemented with recombinant murine CXCL10 (2 g mL −1 , PeproTech) or IgG as control.

Techniques:

Journal: PLOS ONE

Article Title: Extracellular mixed histones are neurotoxic and modulate select neuroimmune responses of glial cells

doi: 10.1371/journal.pone.0298748

Figure Lengend Snippet: Effects of MH on the secretion of glutamate (A), NO (B), CXCL10 (C), and TNF (D) by unstimulated and stimulated BV-2 murine microglia. Cells were exposed to MH (2–50 μg/ml) on their own, or cells were also stimulated 15 min later with either LPS (400 ng/ml), IFN-γ (150 U/ml), or a combination of LPS (20 ng/ml) plus IFN-γ (150 U/ml). Following a 24 h incubation period, concentrations of glutamate in cell-free supernatants were measured by an enzyme-based assay (A); concentrations of nitrite in cell-free supernatants were measured by the Griess assay (B); and concentrations of CXCL10 (C) and TNF (D) were measured by ELISAs. Data (means ± SEM) from four to six independent experiments performed on separate days are presented. * P < 0.05 according to Dunnett’s post-hoc test. P and F values for the one-way randomized block ANOVA are displayed, and the detection limit of each assay is indicated by a dotted line.

Article Snippet: Human and murine recombinant interferon (IFN)-γ, and enzyme-linked immunosorbent assay (ELISA) development kits for human TNF, and both human and murine CXCL10 were acquired from Peprotech (Embrun, ON, Canada).

Techniques: Incubation, Enzymatic Assay, Griess Assay, Blocking Assay

Journal: Bioengineering & Translational Medicine

Article Title: Versatile tissue‐injectable hydrogels capable of the extended hydrolytic release of bioactive protein therapeutics

doi: 10.1002/btm2.10668

Figure Lengend Snippet: Hydrogel‐mediated chemokine gradients sustained via azidoester hydrolysis (A) This illustration depicts the structure of a CCL2‐STEPL fusion protein expressed via the STEPL system in E. coli. ⁹² , ⁹⁷ , ⁹⁸ , ⁹⁹ , ¹⁰⁰ , ¹⁰¹ , ¹⁰² In this variant of sortagging, the protein of interest and sortase are co‐expressed on the same plasmid. (B) This illustration represents the STEPL process as follows: Purified fusion proteins are initially bound to a Ni‐NTA column. Upon adding PolyG‐azidoester and calcium, sortase catalyzes the simultaneous removal of CCL2 and the attachment of PolyG‐4Azidoester, resulting in CCL2‐4azidoester. This product can be collected in the flowthrough, while the remaining fusion protein continues to be bound to the Ni‐NTA column until elution. ¹⁰³ (C) This graph displays month‐long release profiles of CCL2‐4azidoester from a hydrogel in PBS at 37°C, quantified as μg/mL by A 280 readings. Data are presented as mean ± SD for n = 3 replicates. Statistical significance was determined by one‐way ANOVA followed by Tukey's multiple comparison test. (D) In this graph, fold change of CCL2‐4azidoester release over 4 weeks is compared to that of DEAC‐4azidoester. Despite CCL2's much larger size, we observed no significant difference in release rates between the two species. Data are presented as mean ± SE for n = 3 replicates. Statistical significance was determined by multiple unpaired t ‐tests followed by Holm–Šídák post‐hoc correction. [(ns) not significant, (*) p < 0.05.]

Article Snippet: Half of the mice were injected intratumorally every other day with murine CXCL10 in PBS (PeproTech, Cranbury, NJ) or a PBS vehicle control.

Techniques: Variant Assay, Plasmid Preparation, Purification, Comparison