Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 1. Binding and in vitro activity of murine 18V4F hybridoma antibody. (A) ELISA binding of original 18V4F hybridoma antibody to a panel of chemokines (determined in triplicate, shown as mean +/2 standard deviation). Directly coated chemokines were used here for direct comparisons, however in other experiments CCL3 showed a significantly enhanced signal when biotinylated and coated on streptavidin plates. (B) Chemotaxis inhibition by 18V4F hybridoma antibody of CCR5-transfected Ba/F3 cells to 5 ng/mL of CCL3, CCL4, and CCL5. Data are representative of at least three similar experiments. All chemotaxis data are represented as a percent of maximum migration in the absence of inhibitors and is fit using a standard four parameter dose-response model (GraphPad). doi:10.1371/journal.pone.0043332.g001

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Binding Assay, In Vitro, Activity Assay, Enzyme-linked Immunosorbent Assay, Standard Deviation, Chemotaxis Assay, Inhibition, Transfection, Migration

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 2. Diagram of phage display selection strategy. Individual CDR libraries were sequentially panned against CCL3, CCL4 and CCL5. In step 1, phage libraries were combined with biotinylated CCL3 and bound to streptavidin beads. Bound phage were eluted, amplified, and subjected to panning against biotinylated CCL4 and CCL5 in steps 2 and 3, respectively. This process was repeated 4–5 times with increasing stringency to yield sequences with improved affinities. doi:10.1371/journal.pone.0043332.g002

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Selection, Amplification

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 3. Chemotaxis inhibition by affinity matured 18V4F variants. Chemotaxis inhibition by humanized 18V4F Fab, d5 variant, d7 variant, d5d7, and a negative control Fab of CCR5-transfected Ba/F3 cells to 5 ng/mL of (A) CCL3, (B) CCL4, and (C) CCL5. Data are representative of at least two similar experiments. A loss in potency of humanized 18V4F Fab was observed compared with the 18V4F hybridoma shown in Figure 1b and is likely a result of both the humanization process and loss in avidity caused by switching from full IgG to Fab fragment. doi:10.1371/journal.pone.0043332.g003

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Chemotaxis Assay, Inhibition, Variant Assay, Negative Control, Transfection

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 4. Comparison of vCCI and d5d7 binding epitopes. Competitive binding ELISA examining molecules that can disrupt the d5d7-CCL3 binding interaction using d5d7 as a homologous competitor and vCCI-Fc, commercial anti-CCL3 antibody, and control IgG as heterologous competitors. Data are representative of at least two similar experiments. Competition experiments were also completed to analyze the d5d7-CCL4 and d5d7-CCL5 binding interactions and similar binding competition was observed between d5d7 and vCCI-Fc (data not shown). doi:10.1371/journal.pone.0043332.g004

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Comparison, Binding Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 5. Inhibition of chemotaxis induced with mixtures of chemokines by MAb d5d7. Inhibition of chemotaxis of (A) CCR5 transfectants to a pool of recombinant CCL3, CCL4, and CCL5 and (B) CCR1 transfectants to a pool of CCL3 and CCL5, by MAb d5d7 antibody, vCCI-Fc, individual commercial anti-chemokine antibodies (anti-CCL3, anti-CCL4, and anti-CCL5), and IgG controls. Chosen chemokine concentrations were those that produced 50% maximal chemotaxis when tested individually (a pool of 3 ng/mL CCL3, 10 ng/mL CCL4, and 3 ng/mL CCL5 was used in CCR5 experiments and a mixture of 20 ng/ mL CCL3 and 5 ng/mL CCL5 was used in CCR1 experiments). doi:10.1371/journal.pone.0043332.g005

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Inhibition, Chemotaxis Assay, Recombinant, Produced

Journal: PloS one

Article Title: A novel highly potent therapeutic antibody neutralizes multiple human chemokines and mimics viral immune modulation.

doi: 10.1371/journal.pone.0043332

Figure Lengend Snippet: Figure 8. SCID-hu mouse model of leukocyte migration. (A) NSG (NOD/SCID/IL2r-cnull) mice were injected i.v. with human PBMC and allowed to engraft for 10 d. MAb d5d7 was administered i.v. just before chemokines were injected s.c. in Matrigel. After 7 d the skin sites were harvested and single cell suspensions were generated. Human leukocytes were tagged with specific antibodies and analyzed by flow cytometry. (B) Inhibition by MAb d5d7 of skin leukocyte migration into chemokine-embedded Matrigel plugs in NSG mice engrafted with human PBMC. The negative control group consisted of animals treated with s.c. injection of Matrigel + PBS and i.v. administration of control IgG. All other groups had s.c. injections of Matrigel containing CCL3, CCL4, and CCL5 (400 ng each) with i.v. administration of PBS, control IgG, or MAb d5d7 antibody. Data were analyzed using a student t test. doi:10.1371/journal.pone.0043332.g008

Article Snippet: Control inhibitors included vCCI-Fc (produced at VLST – the Fc is from human IgG1 with mutations to prevent interactions with Fc receptors [38]) and commercial antibodies against CCL3 (R&D Systems #MAB270), CCL4 (R&D Systems #MAB271), and CCL5 (R&D Systems #MAB278) as well as IgG controls.

Techniques: Migration, Injection, Generated, Flow Cytometry, Inhibition, Negative Control, Control

Journal: Immunity

Article Title: Single cell RNA sequencing of microglia throughout the mouse lifespan and in the injured brain reveals complex cell-state changes

doi: 10.1016/j.immuni.2018.11.004

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: anti-Tmem119 (for mouse staining, Abcam ab209064, 1:500), anti-Tmem119 (for human staining, Sigma, HPA051870 , 1:500), anti-CCL4/MIP-1 beta (for mouse staining, R&D Systems AF-451 1:25), anti-CCL4/MIP-1 beta (for human staining, R&D Systems AF-271, 1:25), anti-HLA-DR (LN3) (Abcam).

Techniques: Staining, Virus, Control, Recombinant, Multiplex Assay, RNAscope, Gene Expression, Software, Plasmid Preparation

Journal: Cell death and differentiation

Article Title: A keratinocyte-adipocyte signaling loop is reprogrammed by loss of BTG3 to augment skin carcinogenesis.

doi: 10.1038/s41418-024-01304-7

Figure Lengend Snippet: Fig. 2 BTG3-KO keratinocytes release IL1α, IL10, and CCL4 to promote 3T3-L1 adipogenic differentiation. a Schematic workflow of cytokine antibody array analysis. CM was collected after 48 h of culture, and analyzed using a human cytokine antibody array. BTG3-KO CM was a mixture from the three KO clones indicated. b The levels of factors and cytokines, including IL1α, IL10, and CCL4, were increased in BTG3-KO CM. CM from (a) was subjected to antibody array analysis. Signals from duplicated spots were quantified and compared between parental and BTG3-KO cells. CCL7 is shown as an unaltered control. c mRNA expression levels of IL1A, IL10, CCL4, and VEGFD, but not CCL7, were increased in BTG3-KO HaCaT cells. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was performed and results from three independent experiments are shown. d–i Elevated expression levels of IL1α, IL10, and CCL4 in the back skin of Btg3-KO mice. 8-week-old WT and Btg3-KO mice were shaved and their back skin was collected 3 weeks later and embedded for immunohistochemical (IHC) analysis using antibodies against IL1α d, IL10 f, and CCL4 h. Examples of positively stained cell are indicated by arrows. Quantified results (n = 6) are shown in e, g, and i, respectively. j Addition of recombinant IL1α, IL10, and CCL4 to parental CM promotes 3T3-L1 differentiation. Adipogenesis was measured by Oil Red O staining followed by quantification. k Antibody-mediated neutralization of IL1α, IL10 and CCL4 in BTG3-KO CM reduced the effect on 3T3-L1 adipogenic differentiation. *P < 0.05. **P < 0.01.

Article Snippet: The following recombinant proteins were used: IL1α (200-LA-002), IL10 (217-IL-005), CCL4 (271-BME-010), CCL20 (360-MP-025), FGF7 (251-KG-010), and IL15 (247-ILB-005) from R&D Systems, and EGF (E4269) from SigmaAldrich.

Techniques: Ab Array, Clone Assay, Control, Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Immunohistochemical staining, Staining, Recombinant, Neutralization

Journal: Cell death and differentiation

Article Title: A keratinocyte-adipocyte signaling loop is reprogrammed by loss of BTG3 to augment skin carcinogenesis.

doi: 10.1038/s41418-024-01304-7

Figure Lengend Snippet: Fig. 6 Model of BTG3 in the regulation of adipogenesis and the development of skin cancer. Our data are consistent with a role of BTG3 in safeguarding the functional interplay between keratinocytes and adipocytes. In the absence of BTG3, keratinocytes, by releasing IL1α, IL10 and CCL4, promote their own mesenchymal transition by an autocrine mechanism and adipocyte differentiation through paracrine. The latter, in turn, fuels further keratinocyte proliferation and migration by releasing EGF, CCL20, and FGF7, thus forming a feedforward loop to promote skin oncogenesis.

Article Snippet: The following recombinant proteins were used: IL1α (200-LA-002), IL10 (217-IL-005), CCL4 (271-BME-010), CCL20 (360-MP-025), FGF7 (251-KG-010), and IL15 (247-ILB-005) from R&D Systems, and EGF (E4269) from SigmaAldrich.

Techniques: Functional Assay, Migration

Journal: Nature Communications

Article Title: WNT11 Promotes immune evasion and resistance to Anti-PD-1 therapy in liver metastasis

doi: 10.1038/s41467-025-56714-z

Figure Lengend Snippet: a The expression of cytokines was confirmed using qPCR in MC38 and Panc02 scramble, sh Wnt11 , and sh Wnt11 -sh Aff3 cells. b – e ELISA was used to measure the secretion of CXCL10 and CCL4 in the supernatants of MC38 and Panc02 scramble, sh Wnt11 , and sh Wnt11 -sh Aff3 cells. f – h FACS analysis of CD8 + T-cell proliferation (CFSE low) in cocultures with MC38 and Panc02 scramble and sh Wnt11 cells, while CXCR3 and CCR5 were inhibited by AMG487 and maraviroc, respectively, in the coculture system. i – l Cxcl10 and Ccl4 expression were detected using qPCR in MC38 and Panc02 cells with CAMKII knockdown or double knockdown of CAMKII and AFF3. m – o Quantification and representative images of liver weight, number of metastases, and IHC staining of CD8a in an LM mouse model established using MC38 cells treated with a combination of a CXCR3 inhibitor (AMG487) and CCR5 inhibitor (maraviroc). p – r Quantification and representative images of liver weight, number of metastases, and IHC staining of CD8a in an LM mouse model established using Panc02 cells treated with a combination of AMG487 and maraviroc. b – e , g – m , p P -values were calculated using a one-way ANOVA followed by a post hoc Tukey’s multiple comparison test, data are presented as the mean ± SD ( n = 3 biological replicates). Source data are provided as a Source Data file.

Article Snippet: ELISA kits for human CXCL10 (cat. no. 900-T39) and human CCL4 (cat. no. 88-7034-22) were obtained from Thermo Fisher Scientific, Inc., while the mouse CXCL10 (cat. no. DY466-05), mouse CCL4 (cat. no. MMB00), and mouse IL17D ELISA kits (cat. no. DY410) were obtained from R&D Systems, Inc. ELISA kits for human WNT11 (cat. no. LS-F26557-1) and human IL17D (cat. no. LS-F35817-1) were purchased from LSbio.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Knockdown, Immunohistochemistry, Comparison

Journal: Nature Communications

Article Title: WNT11 Promotes immune evasion and resistance to Anti-PD-1 therapy in liver metastasis

doi: 10.1038/s41467-025-56714-z

Figure Lengend Snippet: a , b Anti-PD−1 and CAMKII antagonist KN93 treatment in mice bearing MC38 liver metastases. Tumor progression was monitored post-tumor cell inoculation using live animal bioluminescent imaging. c , d Quantification of the liver weight and number of metastases in the mouse LM model established using MC38 cells in each group at the endpoint. e , f Anti-PD-1 and CAMKII antagonist (KN93) treatment in mice bearing KPC liver metastases. Tumor progression was monitored post-tumor cell inoculation using live animal bioluminescent imaging. g , h Quantification of the liver weight and number of metastases in the mouse LM model established using KPC cells in each group at the endpoint. i , j mIHC staining of I-A/I-E, CD8, CD206, and F4/80 in the sections of MC38 ( i ) and KPC ( j ) liver metastases in each group. k , l The mRNA expression levels of CXCL10, CCL4, and IL17D in tissues of MC38 ( k ) and KPC ( l ) liver metastases in each group. m Representative abdominal MRI images of responder and non-responder to immunotherapy before and after treatment. n – q ELISA analysis of WNT11, CXCL10, CCL4, and IL17D levels in serum samples from responder ( n = 10) and non-responder ( n = 10) mice to immunotherapy. r Schematic representation of the WNT11-mediated immunosuppressive mechanism in liver metastases. b – d , f – h , k , l P -values were calculated using a one-way ANOVA followed by a post hoc Tukey’s multiple comparison test, data are presented as the mean ± SD, n = 5 biological replicates. n – q P -values were calculated using a Student’s t test(two-tailed), in the box plots, bounds of the box spans from 25% to 75% percentile, the line represents the median, and the whiskers show minima and maxima of data points. Source data are provided as a Source Data file.

Article Snippet: ELISA kits for human CXCL10 (cat. no. 900-T39) and human CCL4 (cat. no. 88-7034-22) were obtained from Thermo Fisher Scientific, Inc., while the mouse CXCL10 (cat. no. DY466-05), mouse CCL4 (cat. no. MMB00), and mouse IL17D ELISA kits (cat. no. DY410) were obtained from R&D Systems, Inc. ELISA kits for human WNT11 (cat. no. LS-F26557-1) and human IL17D (cat. no. LS-F35817-1) were purchased from LSbio.

Techniques: Imaging, Staining, Expressing, Enzyme-linked Immunosorbent Assay, Comparison, Two Tailed Test

Journal: NPJ Vaccines

Article Title: Adjuvant composition and delivery route shape immune response quality and protective efficacy of a recombinant vaccine for Entamoeba histolytica

doi: 10.1038/s41541-018-0060-x

Figure Lengend Snippet: Cytokine production from human whole blood stimulated by liposomes containing GLA, 3M-052, both TLR ligands, or neither (empty). Stimulated whole blood was analyzed for production of Th1-associated cytokines a IL-12p70 and b IFN-γ, as well as chemokines c Mip-1β and d MCP-1. The x -axes represent serial dilutions with starting concentrations of 4 µg/ml for GLA and 1.5 µg/ml 3M-052. The y -axes represent concentrations of target analytes secreted post whole blood stimulation with error bars representing the standard error of the mean from three blood donors with each individual value averaged from duplicate wells

Article Snippet: Samples were incubated for 24 h at 37 °C/5% CO 2 in a humidified incubator and plasma supernatants assayed by ELISA for the selected cytokines (Mip-1β [R&D Systems, catalog #DY271]; IL-12p70 [eBioscience, catalog #88-7126-86]; IFN-γ [eBioscience, catalog #88-7316-86]; MCP-1 [eBioscience, catalog #88-7399-88]).

Techniques: Liposomes

Journal: NPJ Vaccines

Article Title: Adjuvant composition and delivery route shape immune response quality and protective efficacy of a recombinant vaccine for Entamoeba histolytica

doi: 10.1038/s41541-018-0060-x

Figure Lengend Snippet: Effect of immunization route on response. Ten mice per group were immunized three times with a 2-week interval between immunizations via intranasal (IN) or subcutaneous (SC) route or a combination of these using GLA-3M-052-PEG2000 liposomes as an adjuvant. Samples were collected 1 week after 3rd immunization. a Stool supernatants were diluted 400-fold to determine LecA-specific IgA titer by ELISA. b Plasma samples were diluted 100,000-fold to determine titers of IgG1 (black circles) and IgG2a (red circles) subtypes. c Splenocytes were restimulated with LecA for 72 h and production of extracellular cytokines in the culture supernatants was determined by Luminex. Cytokine levels of the unstimulated samples were at the baseline (not shown). Error bars represent standard error of the mean. For clarity, statistical significance vs. the adjuvant alone control groups is not represented but is detailed below. For the analysis of the data in plot a , Welch’s one-way ANOVA was employed with Games–Howell correction for multiple comparisons; all of the vaccine groups except for SC + SC + SC were statistically different ( p < 0.05) from the adjuvant alone groups. For the analysis of the data in plot b , data were log-transformed and one-way ANOVA with Tukey’s correction for multiple comparisons was employed; all of the vaccine groups were statistically different ( p < 0.05) from the adjuvant alone groups for both IgG2a and IgG1, and statistically significant differences between vaccine groups represent IgG2a only since no statistical differences were found between vaccine groups for IgG1. For the analysis of the data in plot c , data were log-transformed with a small offset as necessary and Welch’s ANOVA with Games–Howell correction for multiple comparisons was employed (outliers were maintained since they did not affect statistical significance); all vaccine groups were statistically different ( p < 0.05) from the adjuvant alone groups for IFN-γ production, and the adjuvant alone IN + IN + IN was statistically different from the adjuvant alone SC + SC + SC; for IL-17A production, all vaccine groups except for SC + SC + SC were significantly different ( p < 0.05) from the adjuvant alone groups; for IL-2 production, all vaccine groups were significantly different ( p < 0.05) from the adjuvant alone groups

Article Snippet: Samples were incubated for 24 h at 37 °C/5% CO 2 in a humidified incubator and plasma supernatants assayed by ELISA for the selected cytokines (Mip-1β [R&D Systems, catalog #DY271]; IL-12p70 [eBioscience, catalog #88-7126-86]; IFN-γ [eBioscience, catalog #88-7316-86]; MCP-1 [eBioscience, catalog #88-7399-88]).

Techniques: Liposomes, Adjuvant, Enzyme-linked Immunosorbent Assay, Clinical Proteomics, Luminex, Control, Transformation Assay