Journal: EMBO Reports

Article Title: Monomeric agonist peptide/ MHCII complexes activate T‐cells in an autonomous fashion

doi: 10.15252/embr.202357842

Figure Lengend Snippet: Efficient blockade of T‐cell antigen recognition by the 14.4.4 scF V. Shown is the flow cytometric analysis (refer to Appendix Fig A) of antigen‐induced TNF‐α and IFN‐γ expression in 5c.c7 TCR‐transgenic T‐cells co‐cultured with BMDCs which had been prepulsed with MCC peptide antigen (from 0 to 1 μM) in the absence or presence of the 14.4.4 scF V with a representative dot plot (0.05 μM MCC peptide) depicted on the left. Flow cytometry analysis was conducted with n > 11,000 cells per condition. I‐E k is expressed at high numbers on activated CD11c + CD11b + BMDCs. Left panel: staining procedure employed for quantitation. Middle panel: the median number of I‐E k molecules displayed on the surface of non‐activated and activated BMDCs ( n > 10,000 cells per analysis), gated as CD11c + I‐E k+ or CD11c + CD11b + I‐E k hi , as analyzed through quantitative flow cytometry. Statistics: median and Mann–Whitney U ‐test. Data were pooled from two biological replicates each containing n = 2 (BMDCs day 7) and n = 2–4 (activated BMDCs) technical replicates. Right panel: histogram depicting the distribution of surface‐expressed I‐E k molecules on non‐activated (dashed) and activated (solid) CD11c + BMDCs (binning = 0.15 (log scale), n = 14,585 non‐activated BMDCs, n = 24,214 activated BMDCs). Data are representative of two biological replicates. P ‐value format: P > 0.05 (NS), P ≤ 0.01 (**). I‐E k is expressed at high numbers on non‐activated and activated CD19 + B‐cells. Left panel: the median number of I‐E k molecules displayed on the surface of non‐activated and activated CD19 + B‐cells ( n > 10,000 cells per analysis) as analyzed through quantitative flow cytometry. Statistics: median and Mann–Whitney U ‐test. Data were pooled from three biological replicates each containing n = 2–3 technical replicates. Right panel: histogram depicting the distribution of I‐E k molecules expressed on non‐activated and activated CD19 + B‐cells (binning = 0.15 (log scale), n = 14,032 non‐activated CD19 + B‐cells, n = 10,010 activated CD19 + B‐cells). Data are representative of three biological replicates. P ‐value format: P ≤ 0.001 (***). I‐A k and I‐E k molecules are expressed on the surface of activated CD11c + BMDCs and activated CD19 + B‐cells at similar levels. Left panel: staining procedure employed for quantitation. Right panel: I‐A k and I‐E k expression levels as determined on the basis of I‐E k surface expression after correcting for the degree of fluorophore conjugation of the antibodies employed. The mAb titration curves were fitted to a one‐site‐specific binding model to estimate B max and the 95% confidence interval. Goodness of fit R 2 > 0.99. Median fluorescence intensity of n > 10,000 cells shown. These data are representative of one experiment. High I‐E k densities as measured via TIRF microscopy on the surface of activated BMDCs and B‐cells. Left panel: Scheme illustrating 14.4.4 scF V ‐AF647‐based staining as well as interference reflection microscopy (IRM)‐ and total internal reflection fluorescence (TIRF)‐based microscopy readout to confirm attachment and spreading of cells on the ICAM‐1‐coated glass surface and density quantitation, respectively. Middle panel: Example of IRM and TIRF images of indicated cells on ICAM‐1‐functionalized glass slides. Upper scale bar, 10 μm. Lower scale bar, 5 μm. Right panel: Dot plot of I‐E k surface densities on non‐activated BMDCs (median, n = 38 cells), activated BMDCs (median, n = 113 cells), non‐activated B‐cells (median, n = 141 cells), and activated B‐cells (median, n = 188 cells). Data were pooled from two (BMDCs day 7), three (activated BMDCs), and five (non‐activated or activated B‐cells) biological replicates. Statistics: median, unpaired two‐tailed Student's t ‐test. P ‐value format: P ≤ 0.001 (***). Source data are available online for this figure.

Article Snippet: To determine total I‐E k expression levels (14.4.4 scF V ‐AF647 staining under saturating conditions), fluorescence associated with AF647 antibody‐labeled cells was compared to that of AF647 quantification beads (Quantum Alexa Fluor 647 MESF, Bangs Laboratories) serving as reference and measured as a separate sample during the same experiment with the same flow cytometer settings.

Techniques: Expressing, Transgenic Assay, Cell Culture, Flow Cytometry, Staining, Quantitation Assay, MANN-WHITNEY, Conjugation Assay, Titration, Binding Assay, Fluorescence, Microscopy, Two Tailed Test

Journal: EMBO Reports

Article Title: Monomeric agonist peptide/ MHCII complexes activate T‐cells in an autonomous fashion

doi: 10.15252/embr.202357842

Figure Lengend Snippet: A Staining procedure to image the surface distribution of I‐E k on living professional APCs below the diffraction limit of visible light using hsPALM. B BMDCs and B‐cells were stained with 14.4.4 scF V ‐AF647‐biotin and mSav‐cc‐PS‐CFP2 and seeded onto ICAM‐1‐coated glass slides for imaging in TIRF mode. The presence of each cell was verified through acquisition of a white light image (first image, scale bar 10 μm), a single AF647 ensemble fluorescence image (second image), followed by 3,050 image frames displaying single PS‐CFP2 fluorescence events acquired within 7 s to be assembled as a localization map (third image), which was compared to a simulated random I‐E k localization map (fourth image, scale bar 1 μm). Ripley's K analysis was used to compare BMDC‐derived I‐E k distributions (red) or B‐cell‐derived I‐E k distributions (blue) to 10 simulations of randomized distributions with corresponding molecular densities, diffusion parameters, and blinking statistics (gray). Statistics: mean ± standard deviation. C, D Quantification of the surface distribution of (C) CD205 and (D) CD18 on activated BMDCs. Left panels: Staining and imaging procedure. Right panels: BMDCs were stained with the indicated mAbs (NLDC‐145 mAb‐AF647‐biotin or M18/2 mAb‐AF647‐biotin) and mSav‐cc‐PS‐CFP2 for hsPALM imaging. Large scale bars, 10 μm. Small scale bars, 1 μm. Ripley's K analysis was used to compare cell‐derived CD205 distributions (red) to 10 simulations of randomized distributions with corresponding molecular densities, diffusion parameters, and blinking statistics (gray). Statistics: mean ± standard deviation. E Principle of label density variation analysis. A theoretical spatial uncertainty ( σ ) of 35 nm was assigned to each PS‐CFP2 localization and overlapping Gaussian profiles were merged (and intensities were added up) to arrive at three‐dimensional cluster maps ( X , Y position, intensity). Different thresholds were assigned to probe‐derived cluster maps and calculate ρ (the density of localizations in the clustered area μm –2 ) and η (clustered area divided by total area). F Label density variation analysis is consistent with a random surface distribution of I‐E k on activated BMDCs and B‐cells and a clustered surface distribution of CD205 on activated BMDCs which served as a positive control. Quantification of the relative clustered area ( η ) and the density of localizations per clustered area ( ρ ) for indicated molecular species at a threshold of 1.0. The black line indicates the reference curve for a random distribution. This data set was derived from two biological replicates with a total of n = 59 activated BMDCs (I‐E k ), n = 53 activated B‐cells (I‐E k ), and n = 64 activated BMDCs (CD205). Source data are available online for this figure.

Article Snippet: To determine total I‐E k expression levels (14.4.4 scF V ‐AF647 staining under saturating conditions), fluorescence associated with AF647 antibody‐labeled cells was compared to that of AF647 quantification beads (Quantum Alexa Fluor 647 MESF, Bangs Laboratories) serving as reference and measured as a separate sample during the same experiment with the same flow cytometer settings.

Techniques: Staining, Imaging, Fluorescence, Derivative Assay, Diffusion-based Assay, Standard Deviation, Positive Control

Journal: EMBO Reports

Article Title: Monomeric agonist peptide/ MHCII complexes activate T‐cells in an autonomous fashion

doi: 10.15252/embr.202357842

Figure Lengend Snippet: A Scheme illustrating the experimental procedure applied. (i) Activated BMDCs and B‐cells were pulsed with MCC‐PEG 2 ‐biotin. (ii) Resulting I‐E k /MCC‐PEG 2 ‐biotin molecules on the cell surface were blocked with unlabeled mSav after the initial peptide pulse (6–12 h). (iii) Following a recovery lasting 10 min at 37°C, newly arrived and surface‐exposed I‐E k /MCC‐PEG 2 ‐biotin molecules were stained with mSav‐abCAGE635 at room temperature, washed, and (iv) directly subjected to single‐molecule tracking analysis. B To determine the duration of peptide‐loading most adequately for tracking experiments (analysis in C–E), we quantitated the degree of I‐E k ‐peptide loading on activated BMDCs and B‐cells by flow cytometric analysis. Cells were pulsed with MCC‐PEG 2 ‐biotin for up to 36 h and then stained with either 14.4.4 scF V ‐AF647 (reflecting all surface‐resident I‐E k molecules) or mSav‐AF647 (marking I‐E k loaded with MCC‐PEG 2 ‐biotin). Ratios were built from MFIs measured for either scF V ‐AF647‐ or mSav‐AF647‐decorated cells to determine the proportion of surface‐accessible I‐E k molecules loaded with MCC‐PEG 2 ‐biotin. Statistics: mean and standard deviation of two technical replicates. C Activated BMDCs and B‐cells pulsed with MCC‐PEG 2 ‐biotin for 12 and 6 h, respectively, were stained with mSav‐abCAGE635 and seeded on ICAM‐1‐coated glass slides for imaging in TIRF mode at 37°C. Single I‐E k /MCC‐PEG 2 ‐biotin molecules were tracked over time with a time lag of 10.5 ms. The graphs demonstrate the mean square displacement of the total pool and newly arrived I‐E k molecules on activated BMDCs (10,051 trajectories on 14 cells, total pool; 8,607 trajectories on 13 cells, 10 min recovery) and activated B‐cells (11,312 trajectories on 16 cells, total pool; 8,974 trajectories on 15 cells, 10 min recovery). Data are representative of one (BMDCs) and two (B‐cells) biological replicates. Diffusion constants D were calculated based on the first two data points. Fractions were calculated by fitting the first 10 data points. Statistics: mean and standard deviation. D, E Fast‐moving fraction of newly arriving and the total pool of I‐E k molecules on activated BMDCs (D) and activated B‐cells (E) were calculated by fitting the recorded I‐E k trajectories to a binary diffusion model (left panel). Mean square displacement plot of fast‐moving I‐E k molecules (middle panel) and slowly moving I‐E k molecules (right panel) are plotted for the indicated time lags employed in the tracking experiments. Statistics: mean and standard deviation. F = fraction of fast‐moving molecules. D 1 = diffusion constant of fast‐moving fraction. D 2 = diffusion constant of slowly moving fraction. Diffusion constants were calculated by fitting the first two data points. Fractions were calculated by fitting the first 10 data points. Source data are available online for this figure.

Article Snippet: To determine total I‐E k expression levels (14.4.4 scF V ‐AF647 staining under saturating conditions), fluorescence associated with AF647 antibody‐labeled cells was compared to that of AF647 quantification beads (Quantum Alexa Fluor 647 MESF, Bangs Laboratories) serving as reference and measured as a separate sample during the same experiment with the same flow cytometer settings.

Techniques: Staining, Standard Deviation, Imaging, Diffusion-based Assay

Journal: EMBO Reports

Article Title: Monomeric agonist peptide/ MHCII complexes activate T‐cells in an autonomous fashion

doi: 10.15252/embr.202357842

Figure Lengend Snippet: A Principle of TOCCSL to study the stoichiometry of I‐E k on living BMDCs and B‐cells. Cells were quantitatively labeled with the 14.4.4 scF V ‐AF647, placed on an ICAM‐1‐coated glass slide for imaging, and subjected to an illumination sequence of (i) a prebleach image followed by a high‐powered laser pulse to quantitatively ablate the AF647 fluorescence of a particular cell area (as defined by the slit aperture). (ii) Complete photobleaching of AF647 was verified by a control image 100–750 ms after the bleach pulse. (iii) Following a recovery phase of 2–20 s, we next recorded TOCCSL images to detect single diffraction‐limited events resulting from laterally mobile I‐E k molecules, which had moved from the masked into the unmasked area. Finally, the brightness of individual fluorescence events was determined to assess I‐E k ‐complex stoichiometry (shown in B, C). Scale bar, 5 μm. B Quantitative brightness analysis revealed a monomeric contribution of recovered I‐E k molecules on BMDCs ( n = 471 molecules on 59 cells) and B‐cells ( n = 187 molecules on 52 cells). As monomer control, we stained the cells with a cocktail of 14.4.4 scF V ‐AF647‐biotin and 14.4.4 scF V ‐biotin (mixed 1:15) and recorded n = 441 molecules on 50 BMDCs and n = 263 molecules on 57 B‐cells. As dimer control, we stained BMDCs and B‐cells with 14.4.4 scF V ‐AF647‐biotin and mSav‐AF647 which increased the dimeric contribution of recorded fluorescent events to 19% on activated BMDCs ( n = 541 molecules on 64 cells) and 30% on activated B‐cells ( n = 334 molecules on 61 cells). Data are representative of one (B‐cells) and two independent experiments (BMDCs). C TOCCSL measurement of I‐E k ‐bound 14.4.4 scF V ‐AF647‐biotin crosslinked with 125 nM divalent streptavidin on activated BMDCs. n = 301 molecules (monomer control) and n = 294 molecules (dimer control) derived from over 50 cells in one experiment. D Activated B‐cells and BMDCs were stained with a 1:1 premix of the 14.4.4 scF V ‐AF555 and 14.4.4 scF V ‐AF647 and seeded onto ICAM‐1‐coated glass slides for imaging in TIRF mode to probe molecular proximities of APC surface expressed pMHCIIs via FRET imaging. FRET donor intensity (green, background subtracted) was monitored before and after FRET DRAAP. FRET efficiencies were calculated on a pixel‐by‐pixel basis for attached membrane regions (see IRM image). Scale bar, 10 μm. E FRET measurements of I‐E k on activated BMDCs, B‐cells, and PLBs. Activated B‐cells and BMDCs were stained with a 1:1 premix of the 14.4.4 scF V ‐AF555 and 14.4.4 scF V ‐AF647. PLBs featuring I‐E k at indicated densities were decorated with I‐E k /MCC‐AF555 and I‐E k /MCC‐AF647 premixed 1:1. Density‐dependent FRET efficiencies estimated from randomized molecular pMHCII encounters through Monte‐Carlo simulations are shown in gray for FRET donor and acceptors present at a 1:1 ratio. Each dot represents the mean FRET value determined for an individual cell. Error bars represent mean and standard deviation of n = 71 BMDCs, n = 131 B‐cells (donor only), and n = 30–34 PLB positions. Upper and lower boundaries of the gray area represent range for n = 20 simulations per data point. Data were pooled from three (BMDCs) and two (B‐cells) biological replicates. F, G Activated BMDCs were stained with 14.4.4 scF V ‐AF555‐biotin and 14.4.4 scF V ‐AF647‐biotin premixed at a 1:1 ratio. Dimerization of biotinylated scF V s was induced with divalent streptavidin at increasing concentrations and quantified via FRET DRAAP. The addition of monovalent streptavidin (mSav) did not crosslink the 14.4.4‐scF V ‐biotin molecules. Data were pooled from two biological replicates. Statistics: mean and standard deviation and unpaired two‐tailed Student's t ‐test. P ‐value format: P > 0.05 (NS), P ≤ 0.001 (***). Scale bar, 10 μm. Source data are available online for this figure.

Article Snippet: To determine total I‐E k expression levels (14.4.4 scF V ‐AF647 staining under saturating conditions), fluorescence associated with AF647 antibody‐labeled cells was compared to that of AF647 quantification beads (Quantum Alexa Fluor 647 MESF, Bangs Laboratories) serving as reference and measured as a separate sample during the same experiment with the same flow cytometer settings.

Techniques: Labeling, Imaging, Sequencing, Fluorescence, Control, Staining, Derivative Assay, Membrane, Standard Deviation, Two Tailed Test

Journal: EMBO Reports

Article Title: Monomeric agonist peptide/ MHCII complexes activate T‐cells in an autonomous fashion

doi: 10.15252/embr.202357842

Figure Lengend Snippet: Scheme of a FRET‐based assay to visualize TCR‐pMHCII interactions in situ . FRET yields resulting from 5c.c7 TCR‐transgenic T‐cells stained with H57 scF V ‐AF555 and interacting with PLBs featuring I‐E k /MCC‐AF647 as determined via FRET DRAAP and FRET‐sensitized emission. The FRET yield is directly proportional to the TCR occupancy, the ratio of bound TCRs to total TCRs. Scale bar, 5 μm. Synaptic TCR‐ligand engagement was quantified at 25°C via FRET observed for H57 scF V ‐AF555‐decorated 5c.c7 TCR‐transgenic T‐cells engaging PLBs featuring agonist (MCC), weak agonist (T102S), antagonist (K99R), potential co‐agonist (K99A, β2m, ER60), and non‐binding (null) I‐E k molecules displayed at increasing densities. ICAM‐1 and B7‐1 were present at densities of ~100 molecules μm –2 . The TCR occupancy of the entire synapse was calculated according to TCR occupancy = E FRET × 1.21. Statistics: mean and standard deviation of n = 13–81 T‐cell synapses recorded between 2 and 12 min after establishing PLB contact. Data were pooled from 1–2 biological replicates. Scheme of a FRET‐based assay to analyze I‐E k /MCC‐TCR‐binding events in the presence or absence of bystander pMHCIIs with and without the use of a caged peptide (MCC[NVOC]). Relative synaptic TCR occupancy of T‐cells approaching functionalized PLBs displaying the agonist I‐E k /MCC[NVOC]‐AF647 together with I‐E k /K99A‐AF488 as well as ICAM‐1 and B7‐1 (both 100 molecules μm −2 ). Binding of the TCR to I‐E k /MCC‐AF647 was quantitated 25 s after photo‐uncaging via FRET DRAAP. Scale bar, 5 μm. Statistics: mean and standard deviation and unpaired two‐tailed Student's t ‐test. Each gray circle represents a T‐cell synapse ( n = 10–13 cells) of one experiment. P ‐value format: P > 0.05 (NS), P ≤ 0.001 (***). Single‐molecule FRET analysis of T‐cells approaching PLBs functionalized with the photoactivatable agonist I‐E k /MCC[NVOC]‐AF647 in low abundance and I‐E k /K99A‐AF488 in high abundance together with ICAM‐1 and B7‐1 (100 μm −1 ). Ten seconds after the uncaging pulse, single‐molecule FRET events were recorded and counted as individual TCR‐I‐E k /MCC‐binding events (white arrow) if they had disappeared within one frame during recording. Scale bar, 10 μm. Statistics: mean, standard deviation, and unpaired two‐tailed Student's t ‐test. Each gray circle represents a T‐cell synapse ( n = 10–23 cells) of one experiment. P ‐value format: P > 0.05 (NS), P ≤ 0.001 (***). Synaptic TCR occupancy of T‐cells engaging I‐E k /MCC‐AF647 in the presence of increasing densities of I‐E k /K99A‐AF488. Statistics: mean, standard deviation, and unpaired two‐tailed Student's t ‐test. Each gray circle represents a T‐cell synapse ( n = 30–52 cells) of one experiment. P ‐value format: P > 0.05 (NS), P ≤ 0.001 (***). Synaptic TCR occupancy of T‐cells engaging I‐E k /K99A‐AF647 in the presence of increasing I‐E k /MCC‐AF488 densities. Statistics: mean, standard deviation, and unpaired two‐tailed Student's t ‐test. These data are representative of two experiments. Each gray circle represents a T‐cell synapse ( n = 46–61 cells) of one experiment. P ‐value format: P > 0.05 (NS), P ≤ 0.05 (*), P ≤ 0.01 (**), P ≤ 0.001 (***). Synaptic lifetime of I‐E k /MCC‐TCR interactions in the presence or absence of 1,060 I‐E k /K99A‐AF488 μm −2 as measured via single‐molecule FRET imaging at 26°C. Number of smFRET traces ( n = 135–227) to calculate the apparent lifetime (app t off ) for each time lag (56, 84, 252, 476, 924, and 1,820 ms) are summarized in Appendix Fig E. Source data are available online for this figure.

Article Snippet: To determine total I‐E k expression levels (14.4.4 scF V ‐AF647 staining under saturating conditions), fluorescence associated with AF647 antibody‐labeled cells was compared to that of AF647 quantification beads (Quantum Alexa Fluor 647 MESF, Bangs Laboratories) serving as reference and measured as a separate sample during the same experiment with the same flow cytometer settings.

Techniques: In Situ, Transgenic Assay, Staining, Binding Assay, Standard Deviation, Two Tailed Test, Imaging

Journal: EMBO Reports

Article Title: Monomeric agonist peptide/ MHCII complexes activate T‐cells in an autonomous fashion

doi: 10.15252/embr.202357842

Figure Lengend Snippet:

Article Snippet: To determine total I‐E k expression levels (14.4.4 scF V ‐AF647 staining under saturating conditions), fluorescence associated with AF647 antibody‐labeled cells was compared to that of AF647 quantification beads (Quantum Alexa Fluor 647 MESF, Bangs Laboratories) serving as reference and measured as a separate sample during the same experiment with the same flow cytometer settings.

Techniques: Recombinant, Sequencing, Software, Imaging, Isolation, Mutagenesis, Avidin-Biotin Assay, Clinical Proteomics

Journal:

Article Title: Quantitative Expression and Virus Transmission Analysis of DC-SIGN on Monocyte-Derived Dendritic Cells

doi: 10.1128/JVI.76.18.9135-9142.2002

Figure Lengend Snippet: Quantitative FACS measurements of DC-SIGN on MDDCs. Monocytes were purified from peripheral blood mononuclear cells by discontinuous Percoll gradient centrifugation, and MDDCs were derived by GMCSF and IL-4 treatment for 7 days (see Materials and Methods). MDDCs were obtained from seven donors three to five times each over a period of 3 months and phenotyped, i.e., MDDCs were HLA-DRhigh CD11chigh CD14− CD80+ CD83− (data not shown). For each batch of MDDCs we quantified the surface level of DC-SIGN using PE-coupled DC11 (4) in conjunction with the Quantum Simply Cellular microbead kit (Sigma). Shown are the numbers of ABS per cell for each individual (A to G). Each measuring is represented by a symbol, and a horizontal bar shows the average number of ABS for each individual. Because of the technical characteristics of the quantification (see the text), numbers above 250,000 ABS/cell should be considered with caution.

Article Snippet: For quantification we used the Quantum Simply Cellular microbead kit from Sigma according to the manufacturer's instructions and as previously described ( 24 , 30 ).

Techniques: Purification, Gradient Centrifugation, Derivative Assay