Journal: Gene therapy

Article Title: Generation of multi-functional antigen-specific human T-cells by lentiviral TCR gene transfer.

doi: 10.1038/gt.2010.4

Figure Lengend Snippet: Figure 4 Cytokine-stimulated, TCR-transduced human T-cells maintain CD28 and CD62L expression. CD8+ sorted T-cells were transduced with the lentiviral WT1-TCR after activation with OKT3+IL2, IL2 alone, IL7 alone, IL15 alone, IL15+IL7, IL15+IL21, IL7+IL21 and IL2+IL21 (a). The freshly transduced T-cell populations were then re-stimulated with T2 cells loaded with the specific (pWT126) peptide and irradiated feeder cells in the presence of the same cytokine/s used for transduction. After two rounds of peptide/cytokine stimulation the transduced T- cells were sorted into a Vb2.1-positive population using phycoerythrin-labelled anti-Vb2.1 antibodies and anti-phycoerythrin beads (Miltenyi Biotech, Germany). The purified CD8+ Vb2.1+ T-cells were stimulated for an additional 9 days with pWT126 following which expression of CD28 and CD62L was analysed by FACS. These experiments were representative of three independent experiments. (b) The mean fold increase in the mean fluorescence intensity of CD28 expression following transduction in the presence of IL15 alone or IL15+IL21 as compared with OKT3+IL2 is shown. Statistical significance was determined as Po0.05 using a two-tailed t-test. FACS, fluorescence-activated cell sorting; IL, interleukin; TCR, T-cell receptor.

Article Snippet: The concentration of common g-chain cytokines used either alone or in combination as stated was as follows: 20 U ml 1 of human recombinant IL2 (Roche, Basel, Switzerland), 5 ng ml 1 human recombinant IL7 (R&D Systems, Abingdon, Oxfordshire, UK), 10 ng ml 1 human recombinant IL15 (R&D Systems) and 30 ng ml 1 mouse recombinant IL21 (R&D Systems).

Techniques: Expressing, Transduction, Activation Assay, Irradiation, Two Tailed Test, FACS

Journal: Sensors (Basel, Switzerland)

Article Title: Nanofluidic-Based Accumulation of Antigens for Miniaturized Immunoassay

doi: 10.3390/s20061615

Figure Lengend Snippet: Antigen-antibody recognition mechanism and biosensing experiments. ( a ) Positive test: target biotinylated IL10 antigens in solution recognize and bind to the probe antibodies anchored to the surface; fluorescent avidin binds to the biotin, and it is detected by fluorescence microscopy; ( b ) negative test: biotinylated not-matching antigens do not bind with the probe antibodies, and they are washed away, no fluorescence signal can be detected. ( c ) Epifluorescence microscopy images of the funnel after a positive test performed with a biotinylated IL10 concentration of 1.2 pg/mL (right) and after a negative test (left).

Article Snippet: In particular, we prepared solutions of matching antigens consisting of biotinylated recombinant human (rh) IL10 antigens (NF100 kit Fluorokine ® Biotinylated Human IL10, R&D system) diluted in PBS 1X at different concentrations (1.2 pg/mL, 12 pg/mL and 120 pg/mL) and of not-matching antigens, i.e., the negative control reagent provided by the NF100 kit (the soybean trypsin inhibitor biotinylated to the same degree as the cytokine).

Techniques: Avidin-Biotin Assay, Fluorescence, Microscopy, Epifluorescence Microscopy, Concentration Assay

Journal: Nature Medicine

Article Title: BCMA-directed mRNA CAR-T cell therapy for myasthenia gravis: exploratory biomarker analysis of a placebo-controlled phase 2b trial

doi: 10.1038/s41591-025-04170-z

Figure Lengend Snippet: a – e , Change in CD19 + B cells ( a ), CD20 + B cells ( b ), T cells ( c ), natural killer (NK) cells ( d ) and monocytes ( e ) as measured by flow cytometry at month 3 in placebo patients ( n = 10), at month 3 in DC-08 patients ( n = 11) and at month 12 in DC-08 patients ( n = 7) compared to SCR. f – h , Change in immunoglobulin G ( f ), immunoglobulin A ( g ) and immunoglobulin M ( h ) titers at month 3 in placebo patients ( n = 15), at month 3 in DC-08 patients ( n = 19) and at month 12 in DC-08 patients ( n = 13) compared to SCR. i – m , Change in anti-measles ( i ), anti-mumps ( j ), anti-rubella ( k ), anti-diphtheria ( l ) and anti-tetanus ( m ) vaccine titers at month 3 in placebo patients ( n = 15), at month 3 in DC-08 patients ( n = 19) and at month 12 in DC-08 patients ( n = 13) compared to SCR. n – ab , Change in abundance of serum factors analyzed by Olink Target 96 Inflammation panel and expressed as difference in log 2 -scaled NPX between indicated timepoint and respective SCR sample. n , Change in abundance of significantly changed inflammatory factors, along with additional analytes of interest at day 1 ( n = 19). Significant changes in LTA ( P < 0.001), β-NGF ( P = 0.006), TGF-β1 ( P < 0.001), CD6 ( P = 0.010) and IL-7 ( P = 0.018) occurred after leukapheresis ( n = 19). o – t , Change in abundance of inflammatory factors, including IL-6 ( o ), IL-24 ( p ), CCL19 ( q ), ARTN ( r ), RANKL ( s ) and VEGFA ( t ), after treatment with DC-08 compared to month 3 in placebo patients ( n = 10) and DC-08 patients at month 1 ( n = 11), month 3 ( n = 11) and month 12 ( n = 7). Decreases in IL-6 ( P = 0.019) and IL-24 ( P = 0.008) occurred at month 3 in DC-08-treated patients relative to placebo ( o , p ). u – x , Change in abundance of inflammatory cytokines, including IL-6 ( u ), CCL19 ( v ), RANKL ( w ) and VEGFA ( x ), in the DC-08 patient cohort between clinical responders at month 1 and month 3 ( n = 6) and non-responders at month 1 and month 3 ( n = 5). RANKL was significantly reduced in responders at month 1 ( P = 0.035) and month 3 ( P = 0.030) ( w ). y – ab , Change in abundance of inflammatory cytokines, including IL-6 ( y ), CCL19 ( z ), RANKL ( aa ) and VEGFA ( ab ), in the DC-08 patient cohort between patients who had not received prior treatment with biologics (that is, complement inhibitors, FcRn inhibitors or CD19/CD20-targeting antibodies) at month 1 and month 3 ( n = 5) and patients who had received prior biologics at month 1 and month 3 ( n = 6). IL-6 ( P = 0.010), CCL19 ( P = 0.002) and VEGFA ( P < 0.001) were significantly decreased at month 1 in patients who had not received biologics prior to DC-08 ( y – ab ). Additionally, VEGFA was significantly decreased in patients who had not received biologics prior to DC-08 at month 3 ( P = 0.003). For lymphocyte subsets, immunoglobulin titers and vaccine titers, P values were generated with a two-sided unpaired t -test for each comparison between treatment groups and a paired two-sided t -test between longitudinal timepoints within each treatment group. For cytokine analysis, P values were generated using a linear mixed-effects model. Box-and-whisker plots show the median, interquartile range and full range of data, along with individual data points. Source data are provided for this figure. NS, not significant.

Article Snippet: Recombinant human IL-7 and IL-15 (Proteintech) were added at 100 ng ml −1 each, and 25 ng ml −1 OKT3 (Takara Bio, anti-CD3 monoclonal antibody) was added on culture day 0 to initiate T cell activation and expansion.

Techniques: Flow Cytometry, Generated, Comparison, Whisker Assay