Journal: Scientific Reports

Article Title: Determining potential immunomodulatory drug efficacy in sepsis using ELISpot

doi: 10.1038/s41598-025-92016-6

Figure Lengend Snippet: Adjuvant effect on IFN-γ production in conjunction with anti-CD3/CD28 mAb stimulation. Samples were stimulated with anti-CD3/28 mAb with and without either anti-PD-1 mAb (Nivolumab; NIVO) or IL-7. Anti-CD/CD28 mAb (CD3) increased IFN-γ SFU (upper panels), SS (upper-middle panels) and TWI (lower-middle panels) in samples from both Healthy donors (left, n = 46) and Septic patients (right, n = 63) as compared to unstimulated spontaneous (SPON) production. NIVO did not increase CD3 induced IFN-γ SFU, SS, or TWI, while IL-7 did increase CD3 induced IFN-γ SFU, SS, and TWI. SFU (Top panels) were normalized to the number of lymphocytes plated in the ELISpot well (Bottom panels, n = 34 healthy and n = 59 septic). Lymphocyte number per well was determined by multiplying the draw specific ALC (K/cumm) by 1000 (converting K/cumm to cells/cumm) then by the volume of blood added to the well (5µL). Pairwise statistical relationships of SFU/Lymphocyte remain largely the same as SFU. Red bars represent Median with Interquartile Ranges (IQR) *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by Friedman Test with multiple comparisons.

Article Snippet: Immunomodulatory agents included LPS (1 ng/mL, ENZO Life Sciences, Farmingdale, NY, USA), anti-CD3 and anti-CD28 mAb (0.5 and 5 ug/mL respectively, or 0.125 and 1.25 ng/mL for low dose, BioLegend, San Diego, CA, USA), recombinant human IL-7 (50 ng/mL, R&D Systems, Minneapolis, MN, USA), anti-PD1 mAb (1 ug/mL, Nivolumab, Bristol Myers Squibb, New York, NY, USA), Dexamethasone (1 ug/mL, Viatris, formerly Mylan, Canonsburg, PA, USA), and Resiquimod (1 ug/mL, Invivogen, San Diego, CA, USA), using IFN-γ and TNF-α ELISpot kits (CTL).

Techniques: Adjuvant, Enzyme-linked Immunospot

Journal: Scientific Reports

Article Title: Determining potential immunomodulatory drug efficacy in sepsis using ELISpot

doi: 10.1038/s41598-025-92016-6

Figure Lengend Snippet: Difference in relative IFN-γ response to IL-7 between healthy donors and septic patients. A. IL-sevenfold-changes were calculated for SFU, SS, and TWI for all samples; anti-CD3/CD28 mAb (CD3) + IL-7 values were divided by CD3 alone to yield fold-change. Samples from healthy donors displayed a higher responsiveness to IL-7 in the SFU (Left) and TWI (Right) measurements, based on fold-change, than did those from septic patients. B. Visualization of paired data points for CD3 alone or with IL-7 for samples from healthy donors (Left, n = 46) and septic patients (Right, n = 63). The bulk of samples show an increase of SFU and SS with IL-7 treatment. Red bars represent Median with IQR. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by ( A. ) Mann–Whitney or ( B. ) Wilcoxon tests.

Article Snippet: Immunomodulatory agents included LPS (1 ng/mL, ENZO Life Sciences, Farmingdale, NY, USA), anti-CD3 and anti-CD28 mAb (0.5 and 5 ug/mL respectively, or 0.125 and 1.25 ng/mL for low dose, BioLegend, San Diego, CA, USA), recombinant human IL-7 (50 ng/mL, R&D Systems, Minneapolis, MN, USA), anti-PD1 mAb (1 ug/mL, Nivolumab, Bristol Myers Squibb, New York, NY, USA), Dexamethasone (1 ug/mL, Viatris, formerly Mylan, Canonsburg, PA, USA), and Resiquimod (1 ug/mL, Invivogen, San Diego, CA, USA), using IFN-γ and TNF-α ELISpot kits (CTL).

Techniques: MANN-WHITNEY

Journal: Scientific Reports

Article Title: Determining potential immunomodulatory drug efficacy in sepsis using ELISpot

doi: 10.1038/s41598-025-92016-6

Figure Lengend Snippet: Septic patient samples produce greater IFN-γ SFU and SFU/Lymphocyte than Healthy donor samples. Comparison between Healthy donor (n = 46) and Septic patient (n = 63) whole blood samples shows septic blood samples have higher IFN-γ SFU (top) than healthy donor samples in response to anti-CD3/CD28 mAb (CD3) alone (left), CD3 + anti-PD-1 mAb (Nivolumab; NIVO) (middle), and CD3 + IL-7 (right). To account for high variability in cell counts among septic patients and between septic and healthy samples, SFU were normalized to number of lymphocytes plated in ELISpot wells (bottom, n = 34 healthy and n = 59 septic). When SFU were normalized to lymphocyte numbers, the differences between septic and healthy IFN-γ production became more apparent. As no significant differences were discerned between CD3 treated samples and CD3 + NIVO treated samples, the differences displayed in the “CD3 + NIVO” column between healthy and septic are a consequence of the greater responsiveness of septic samples to CD3 treatment. Red bars represent Median with IQR. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by Mann–Whitney test.

Article Snippet: Immunomodulatory agents included LPS (1 ng/mL, ENZO Life Sciences, Farmingdale, NY, USA), anti-CD3 and anti-CD28 mAb (0.5 and 5 ug/mL respectively, or 0.125 and 1.25 ng/mL for low dose, BioLegend, San Diego, CA, USA), recombinant human IL-7 (50 ng/mL, R&D Systems, Minneapolis, MN, USA), anti-PD1 mAb (1 ug/mL, Nivolumab, Bristol Myers Squibb, New York, NY, USA), Dexamethasone (1 ug/mL, Viatris, formerly Mylan, Canonsburg, PA, USA), and Resiquimod (1 ug/mL, Invivogen, San Diego, CA, USA), using IFN-γ and TNF-α ELISpot kits (CTL).

Techniques: Comparison, Enzyme-linked Immunospot, MANN-WHITNEY

Journal: Scientific Reports

Article Title: Determining potential immunomodulatory drug efficacy in sepsis using ELISpot

doi: 10.1038/s41598-025-92016-6

Figure Lengend Snippet: Effect of dexamethasone on IFN-γ production. IFN-γ SFU (Top), SS (Middle), and TWI (Bottom), are reduced when dexamethasone (DEX) is added to ELISpot. DEX abrogates anti-CD3/CD28 mAb (CD3) induced IFN-γ production. DEX-induced reduction of IFN-γ production is still present when co-cultured with anti-CD3/CD28 mAb and IL-7, though there is still an IL-7 effect present even with DEX co-treatment. Results were similar in healthy donor (n = 46) and septic patient (n = 63) samples. Red bars represent Median with IQR. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 as determined by Friedman Test with multiple comparisons.

Article Snippet: Immunomodulatory agents included LPS (1 ng/mL, ENZO Life Sciences, Farmingdale, NY, USA), anti-CD3 and anti-CD28 mAb (0.5 and 5 ug/mL respectively, or 0.125 and 1.25 ng/mL for low dose, BioLegend, San Diego, CA, USA), recombinant human IL-7 (50 ng/mL, R&D Systems, Minneapolis, MN, USA), anti-PD1 mAb (1 ug/mL, Nivolumab, Bristol Myers Squibb, New York, NY, USA), Dexamethasone (1 ug/mL, Viatris, formerly Mylan, Canonsburg, PA, USA), and Resiquimod (1 ug/mL, Invivogen, San Diego, CA, USA), using IFN-γ and TNF-α ELISpot kits (CTL).

Techniques: Enzyme-linked Immunospot, Cell Culture

Journal: ACS Omega

Article Title: An AviTag/BirA Platform for IL7: Minimized Activity Loss over Random Biotinylation

doi: 10.1021/acsomega.5c12146

Figure Lengend Snippet: Schematic of a DNA sequence map of hIL7 expression vectors.

Article Snippet: Transferred membranes were blocked with 5% (w/v) skimmed milk in TBST for 1 h at room temperature, then probed overnight at 4 °C with anti-hIL7 antibody (Abbkine) and HRP-conjugated streptavidin (SA-HRP, Beyotime Biotechnology).

Techniques: Sequencing, Expressing

Journal: ACS Omega

Article Title: An AviTag/BirA Platform for IL7: Minimized Activity Loss over Random Biotinylation

doi: 10.1021/acsomega.5c12146

Figure Lengend Snippet: Purification and identification of the fusion protein. (a) Transformation of the recombinant plasmid into competent cells, plated on LB solid medium supplemented with ampicillin. (b) SDS-PAGE with Coomassie blue staining of proteins extracted from recombinant bacteria cultured in LB liquid medium containing ampicillin at 37 °C and 200 rpm under different culture durations (4, 8, 12 h) and different IPTG concentrations (0.1, 0.5, 1.0 mM). (c) Analysis of protein expression under uninduced conditions and after induction (12 h, 0.5 mM IPTG) in LB liquid medium with ampicillin at 37 °C and 200 rpm. Samples include uninduced, pellet, supernatant, flow-through, and elution fractions with 300 mM imidazole. The right panel shows the purification of soluble proteins and Western blot detection using an anti-His antibody. (d) SDS-PAGE with Coomassie blue staining of soluble and inclusion body proteins. Lanes from left to right represent pellet, supernatant, flow-through, and elution fractions with different imidazole concentrations. (E)­Western blot analysis of soluble and inclusion body proteins using anti-hIL7 and antibiotin antibodies is also shown. Note: the following terms are used throughout the experimental procedures. Pellet: solid fraction obtained after centrifugation. Supernatant: liquid fraction obtained after centrifugation. Flow-through: fraction that passes through the His-tag nickel affinity column during sample loading. Washing: fraction eluted with a low concentration imidazole (or ionic) buffer to remove nonspecifically bound impurities. Elution: fraction containing target protein, eluted with high concentration imidazole (or ionic) buffer for competitive displacement. Concentration notation: mM = millimolar concentration. Notation 300 mM-1 refers to first elution step performed with 300 mM imidazole and subsequent elutions are labeled accordingly (e.g., 300 mM-2, 300 mM-3).

Article Snippet: Transferred membranes were blocked with 5% (w/v) skimmed milk in TBST for 1 h at room temperature, then probed overnight at 4 °C with anti-hIL7 antibody (Abbkine) and HRP-conjugated streptavidin (SA-HRP, Beyotime Biotechnology).

Techniques: Purification, Transformation Assay, Recombinant, Plasmid Preparation, SDS Page, Staining, Bacteria, Cell Culture, Expressing, Western Blot, Centrifugation, Affinity Column, Concentration Assay, Labeling

Journal: ACS Omega

Article Title: An AviTag/BirA Platform for IL7: Minimized Activity Loss over Random Biotinylation

doi: 10.1021/acsomega.5c12146

Figure Lengend Snippet: Extraction and stepwise elution of inclusion body proteins. (a) Western blot analysis of fractions from the solubilization and washing of inclusion bodies under pH 4.6 or 250 mM imidazole conditions. Lanes (left to right): supernatant, flow-through, wash, and elute fractions for each condition. The membrane was probed with anti-hIL7 and antibiotin antibodies. (b) Coomassie blue-stained SDS-PAGE analysis of proteins during the washing and elution processes. Lanes show the supernatant, flow-through, and sequentially washed fractions by low concentration gradients of imidazole (six washes each at 10, 20, and 30 mM imidazole), followed by ten consecutive elutions using 250 mM imidazole. Note that “mM” indicates millimolar; “10 mM-2” is the second wash with 10 mM imidazole, and subsequent steps are labeled in the same manner.

Article Snippet: Transferred membranes were blocked with 5% (w/v) skimmed milk in TBST for 1 h at room temperature, then probed overnight at 4 °C with anti-hIL7 antibody (Abbkine) and HRP-conjugated streptavidin (SA-HRP, Beyotime Biotechnology).

Techniques: Extraction, Western Blot, Membrane, Staining, SDS Page, Concentration Assay, Labeling

Journal: ACS Omega

Article Title: An AviTag/BirA Platform for IL7: Minimized Activity Loss over Random Biotinylation

doi: 10.1021/acsomega.5c12146

Figure Lengend Snippet: Functional validation of the fusion protein. (a) Western blot analysis probed with anti-hIL7 antibody and SA-HRP to evaluate biotinylation efficiency in the presence or absence of exogenous biotin supplementation. (b) Analysis of the conjugation between randomly biotinylated Bovine Serum Albumin (BSA) and SA by native page. Lanes represent different concentrations of SA and BSA. Additionally, the conjugation efficiency of site-specifically biotinylated Avi-tagged Biotin-IL15 and the fusion protein biotin-IL7 with SA are compared. Note: um: Micromolar concentration (c) CCK-8 assay assessing the proliferation of preactivated T cells after coculture with the fusion protein that had been desalted and concentrated via ultrafiltration.

Article Snippet: Transferred membranes were blocked with 5% (w/v) skimmed milk in TBST for 1 h at room temperature, then probed overnight at 4 °C with anti-hIL7 antibody (Abbkine) and HRP-conjugated streptavidin (SA-HRP, Beyotime Biotechnology).

Techniques: Functional Assay, Biomarker Discovery, Western Blot, Conjugation Assay, Clear Native PAGE, Concentration Assay, CCK-8 Assay