Journal: Frontiers in Immunology

Article Title: RIG-I and TLR-7/8 agonists as combination adjuvant shapes unique antibody and cellular vaccine responses to seasonal influenza vaccine

doi: 10.3389/fimmu.2022.974016

Figure Lengend Snippet: Source of reagents and kits used in the study.

Article Snippet: Mouse IL-4 ELISPOT kit , RnD systems , EL404.

Techniques: In Vivo, Luciferase, Enzyme-linked Immunospot, Reverse Transcription, Enzyme-linked Immunosorbent Assay, Isolation

Journal: FASEB bioAdvances

Article Title: AJP001, a novel helper T‐cell epitope, induces a humoral immune response with activation of innate immunity when included in a peptide vaccine

doi: 10.1096/fba.2019-00056

Figure Lengend Snippet: Vaccination with AJP001 induces antibody production and T‐cell activation in mice. A. Ang II, a mixture of Ang II and AJP001 or the AJP001‐Ang II conjugate vaccine was administered intracutaneously to 7‐week‐old female BALB/cA three times at 2‐week intervals. The anti‐Ang II IgG antibody titer in serum samples collected at 0, 2, 4, 6, and 8 weeks was detected by ELISA. The data represent the mean OD at 450 nm and the SD at each serum dilution fold (n = 3). B, C. The AJP001‐Ang II conjugate vaccine (AJP001‐Ang II 100 μg) was administered intracutaneously to 7‐week‐old female BALB/cA (B) or 7‐week‐old male C57BL/6 (C) mice three times at 2‐week intervals without any adjuvant cotreatment. The anti‐Ang II IgG antibody titer in serum sample collected at 0, 2, 4, 6, and 8 weeks was detected by ELISA. The data represent the mean OD at 450 nm and the SD at each serum dilution fold (n = 3). D. The AJP001‐Ang II conjugate vaccine was administered intracutaneously to 7‐week‐old female BALB/cA mice at a dose of 20, 100, or 500 μg per mouse three times at 2‐week intervals without any adjuvant cotreatment. The anti‐Ang II IgG antibody titer in serum samples collected at 0, 2, 4, 6, and 8 weeks was measured by ELISA. The titers are expressed as the dilution fold of the serum giving half‐maximal absorbance at 450 nm. All data are expressed as the mean ± SD (n = 3). * P < .05, ** P < .01 and *** P < .001 vs the saline group. E, F. Antigen‐specific activation of T cells in AJP001‐Ang II‐immunized mice was evaluated by an ELISpot assay. Splenocytes were isolated from AJP001‐Ang II‐immunized mice and stimulated with Angiotensin II or AJP001 at a concentration of 10 μg/mL. PMA and ionomycin (100 ng/m each) were added to positive control wells, and medium was added as a negative control. The number of IFN‐γ‐ (E) or IL‐4‐producing (F) cells was detected by counting spots using a stereomicroscope. The number of spots was quantified in the duplicate or triplicate wells of each mouse. The data represent the mean ± SD (n = 3). * P < .05 and ** P < .01 vs the saline group

Article Snippet: Mouse IFN‐γ ELISpot Development Module, Mouse IL‐4 ELISpot Development Module and ELISpot Blue Color Module were obtained from R&D Systems, Inc.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Adjuvant, Saline, Enzyme-linked Immunospot, Isolation, Concentration Assay, Positive Control, Negative Control

Journal: Scientific Reports

Article Title: Gene Set Enrichment Analysis Identifies LIF as a Negative Regulator of Human Th2 Cell Differentiation

doi: 10.1038/srep00464

Figure Lengend Snippet: Panel a shows results of IL-4 ELISPOT experiments. Culture conditions are mentioned at the bottom. Error bars represent standard error of mean and * denotes significant (p value 0.017) difference between the two sample (Two tailed paired sample t test). In panel b blue line represents IL-4 level detected in the supernatant at given time points and red line is a measure of IL-4R expression level at corresponding time points. Panel c shows a result obtained in a CFSE labeling experiment in ERK silenced cells. Red, blue and green colors represent not activated, TCR + IL-4 stimulated and TCR + IL-4 stimulated at day 1 followed by additional doses of IL-4 at day 3,4 and 5. Less fluorescence indicates more cell divisions because as the cells divide fluorescent label is diluted.

Article Snippet: ELISPOT assays for IL-4were performed with human IL-4 ELISPOT kits (R & D systems).

Techniques: Enzyme-linked Immunospot, Two Tailed Test, Expressing, Labeling, Fluorescence

Journal: Scientific Reports

Article Title: Gene Set Enrichment Analysis Identifies LIF as a Negative Regulator of Human Th2 Cell Differentiation

doi: 10.1038/srep00464

Figure Lengend Snippet: Panel a shows mRNA expression profile of STAT3 upstream molecules. Panel b shows ELISPOT results for IL-4 producing cells in differentially activated populations. Panel c shows results obtained in LIF ELISA experiments. Panel d and e are mRNA expression data adapted form microarray of LIF and IL-6 respectively. Fold change is calculated with respect to untreated sample. In all cases mean of three biological and two technical replicates are shown. Error bars represent standard deviation and * represents significance at p value of less than 0.05 (see text).

Article Snippet: ELISPOT assays for IL-4were performed with human IL-4 ELISPOT kits (R & D systems).

Techniques: Expressing, Enzyme-linked Immunospot, Enzyme-linked Immunosorbent Assay, Microarray, Standard Deviation

Journal: International journal of cancer

Article Title: Induction of antigen-specific TH 9 immunity accompanied by mast cell activation blocks tumor cell engraftment.

doi: 10.1002/ijc.30121

Figure Lengend Snippet: Figure 3. Vaccination of CEA.Tg mice with rCEA N domain mixed with poly I:C results in the production of CEA N domain-specific TH9 cells. (a) Enumeration of rCEA-specific IL-2, IL-9, IL-4 and IFN-g spot forming units (DSFUs) from immunized and control mice as measured by cytokine ELISPOT. Histogram bars represent averaged DSFU values measured from two independent immunization trials (n 5 3 per group). The number of Ag-specific cytokine secreting lymphocytes (DSFUs) was calculated by subtracting background values (from wells containing unstimulated cells) from measured values in treated groups. Asterisk denotes statistical significance (p 0.05; Student-t-test) when com- pared with the frequency of CEA-specific cytokine secreting cells derived from non-immunized CEA.Tg mice. (b) Intracellular cytokine stain- ing of IL-9 in CEA–specific CD31 CD41 lymphocytes. Splenocytes from immunized and control mice were stimulated, in vitro, with rCEA N domain (10 mg/mL) for 72 hrs, followed by staining for IL-9 production in TH lymphocyte populations. (c) Comparison of the number of CEA- specific IL-9 producing TH cells between immunized and control mice. * Denotes statistical significance (p 0.05) when compared to non- immunized CEA.Tg mice. Statistical significance was determined using the Student-t test, with Welch’s Correction.

Article Snippet: CEA-specific cytokine secreting cells were quantified using the IFN-g, IL-2, IL-9 and IL-4 ELISPOT development modules (R&D Systems; Minneapolis, MN, USA) according to the manufacturer’s recommendations with the exception that the antibodies and enzyme-coupled streptavidin were diluted at a concentration of 1:100.

Techniques: Control, Enzyme-linked Immunospot, Derivative Assay, Staining, In Vitro, Comparison