T cells sum up intermittent TCR stimulation in vivo.

<p>OT-II T cells stimulated 5 h in vitro or in vivo and rested in a naïve recipient display a proliferative advantage upon subsequent antigen encounter compared to their unstimulated counterparts. A. OT-II T cells were stimulated in vitro for 5 h with anti-CD3/anti-CD28 beads, or left unstimulated, then labeled with CFSE before transfer into wild-type C57BL/6 hosts. A secondary stimulation was provided 20 h later by injection of 50 µg LPS followed by OVA^323–339 peptide (or PBS control). Recipient spleens were harvested 48 hours later to assess transferred cells for proliferation. OT-II T cells that had been prestimulated underwent additional rounds of division, evident by CFSE dilution, as compared to their non-prestimulated counterparts. Graph shows the percentage of recovered T cells that had undergone the indicated number of cell divisions. Results are mean±sem and are representative of 3 independent experiments. B. OT-II T cells were stimulated in vivo for 5 h by injecting LPS±OVA^323–339 peptide into OT-II mice and harvesting LN cells 5 h later. Cells were labeled with CFSE before transfer into wild-type C57BL/6 hosts. A secondary stimulation was provided by injection with LPS±OVA^323–339 peptide 20 h later. Recipient spleens were harvested 48 h later to assess transferred cells for proliferation. Graph shows the percentage of recovered T cells that had undergone the indicated number of cell divisions. Results are mean±sem and are representative of 2 independent experiments.</p

T cells have the ability to sum up periods of sub-optimal stimulation in vitro.

<p>A. Splenocytes were stimulated with anti-CD3/anti-CD28 magnetic beads for intervals of 10 minutes (indicated by grey shading) separated by rest periods of 30 minutes. Shedding of CD62L on CD4 T cells (top panel) and CD8 T cells (bottom panel) occurs exclusively during the periods of incubation with the beads, suggesting a rapid and complete cessation of TCR signaling upon removal of the beads. B. CD4 T cells, when stimulated (grey shading) with anti-CD3/anti-CD28 beads for 5 h on day 1 (D1), demonstrate a memory-like response in induction of the activation markers CD25 (top panel) and CD44 (bottom panel) upon secondary stimulation for 5 h on day 2 (D2). C. Top: OT-I splenic T cells do not divide when stimulated with anti-CD3/anti-CD28 beads for 5 h alone either on day 1 (left panel) or day 2 (middle panel), but commit to proliferation after an additive stimulus of 5 h on day 1 and 5 h on day 2 separated by a 20 h rest period (right panel). Numbers indicate percentages of cells with diluted CFSE. Bottom: combinations of two periods of stimulation adding up to 10 h show additive effects on proliferation. Stimulations of day 1 (D1) and day 2 (D2) were separated by a rest period of 20 h. Results are representative of at least 2 independent experiments.</p

c-fos is rapidly induced and phosphorylated upon T cell stimulation and remains stable hours after stimulus withdrawal.

<p>Kinetics of c-fos total protein and its phosphorylated forms in CD4 T cells during a 2 h stimulation with anti-CD3/anti-CD28 beads and 1 h, 2 h, 3 h or 20 h after removal of beads as determined by intracellular staining and FACS analysis. In order to display data from all 3 stainings in a single graph, results are shown as the percent of the maximum change of fluorescence observed for each antibody, e.g. c-fos MFI of the sample/(max c-fos MFI observed in the time course-baseline c-fos MFI)×100. B. Representative images taken by an imaging flow cytometer of CD4 T cells stained intracellularly for c-fos total protein and DAPI. Cells were either left unstimulated or stimulated for 2 h with anti-CD3/anti-CD28 beads, then harvested immediately or 1 h, 2 h or 3 h after stimulus withdrawal. A positive control sample was stimulated with PMA/ionomycin for 2 h. C. LN cells were stimulated 2 h with anti-CD3/anti-CD28 beads and harvested either immediately or 1 h, 2 h or 3 h after removal of beads. The cells were stained for CD4 and for total c-fos protein (top panel) or its phosphorylated forms, pSer362 (middle panel) or pThr325 (bottom panel) before analysis on an imaging flow cytometer. Colocalization with DAPI was used to determine nuclear localization in CD4+ T cells. Each dot represents a different sample. Results (mean±sem) are shown as the percent change in mean fluorescence intensity (MFI) from baseline levels in unstimulated T cells and are pooled from at least 3 independent experiments.</p

Induction of c-fos is additive during sequential in vivo antigen-specific stimulations.

<p>A. Experimental design: CD45.1 Marilyn TCR transgenic mice were injected with 5 µg Dby peptide and splenocytes and LN cells were harvested 30 minutes post-injection and left unlabeled. In parallel, control Marilyn mice were injected with PBS and splenocytes and LN cells were harvested 30 minutes post-injection and labeled with Cell-Tracker Blue. CD4+ T cells from both sources were purified by negative selection and mixed at a 1∶1 ratio before adoptive transfer into wildtype CD45.2 C57BL/6 congenic mice. Host mice received an injection of 5 µg Dby peptide or PBS, and splenocytes were harvested 30 minutes later for intracellular staining of c-fos followed by FACS analysis. B. Left panel: Upon adoptive transfer, Marilyn T cells can be identified in the host spleen based on CD45.1 expression. Furthermore, Marilyn T cells purified from mice that received peptide (orange box, +Dby) can be distinguished from those purified from mice that did not receive peptide (blue box, +PBS) based on Cell-Tracker Blue labeling (left panel). Right panel: A single stimulation with Dby induces upregulation of c-fos (blue line) but two sequential stimulations result in a further increase in c-fos levels (orange line). Shaded gray histograms show c-fos levels in endogenous CD4 T cells as a baseline comparison. C. Change in of c-fos protein levels in Marilyn T cells at the end of the second in vivo stimulation for the indicated combination of stimulation. Results are compiled from 2 independent experiments and shown as the percentage (mean±sem) of the maximum c-fos fluorescence observed in each experiments.</p

DataSheet_3_Beyond 40 fluorescent probes for deep phenotyping of blood mononuclear cells, using spectral technology.pdf

The analytical capability of flow cytometry is crucial for differentiating the growing number of cell subsets found in human blood. This is important for accurate immunophenotyping of patients with few cells and a large number of parameters to monitor. Here, we present a 43-parameter panel to analyze peripheral blood mononuclear cells from healthy individuals using 41 fluorescence-labelled monoclonal antibodies, an autofluorescent channel, and a viability dye. We demonstrate minimal population distortions that lead to optimized population identification and reproducible results. We have applied an advanced approach in panel design, in selection of sample acquisition parameters and in data analysis. Appropriate autofluorescence identification and integration in the unmixing matrix, allowed for resolution of unspecific signals and increased dimensionality. Addition of one laser without assigned fluorochrome resulted in decreased fluorescence spill over and improved discrimination of cell subsets. It also increased the staining index when autofluorescence was integrated in the matrix. We conclude that spectral flow cytometry is a highly valuable tool for high-end immunophenotyping, and that fine-tuning of major experimental steps is key for taking advantage of its full capacity.</p

DataSheet_5_Beyond 40 fluorescent probes for deep phenotyping of blood mononuclear cells, using spectral technology.pdf

The analytical capability of flow cytometry is crucial for differentiating the growing number of cell subsets found in human blood. This is important for accurate immunophenotyping of patients with few cells and a large number of parameters to monitor. Here, we present a 43-parameter panel to analyze peripheral blood mononuclear cells from healthy individuals using 41 fluorescence-labelled monoclonal antibodies, an autofluorescent channel, and a viability dye. We demonstrate minimal population distortions that lead to optimized population identification and reproducible results. We have applied an advanced approach in panel design, in selection of sample acquisition parameters and in data analysis. Appropriate autofluorescence identification and integration in the unmixing matrix, allowed for resolution of unspecific signals and increased dimensionality. Addition of one laser without assigned fluorochrome resulted in decreased fluorescence spill over and improved discrimination of cell subsets. It also increased the staining index when autofluorescence was integrated in the matrix. We conclude that spectral flow cytometry is a highly valuable tool for high-end immunophenotyping, and that fine-tuning of major experimental steps is key for taking advantage of its full capacity.</p

DataSheet_1_Beyond 40 fluorescent probes for deep phenotyping of blood mononuclear cells, using spectral technology.pdf

The analytical capability of flow cytometry is crucial for differentiating the growing number of cell subsets found in human blood. This is important for accurate immunophenotyping of patients with few cells and a large number of parameters to monitor. Here, we present a 43-parameter panel to analyze peripheral blood mononuclear cells from healthy individuals using 41 fluorescence-labelled monoclonal antibodies, an autofluorescent channel, and a viability dye. We demonstrate minimal population distortions that lead to optimized population identification and reproducible results. We have applied an advanced approach in panel design, in selection of sample acquisition parameters and in data analysis. Appropriate autofluorescence identification and integration in the unmixing matrix, allowed for resolution of unspecific signals and increased dimensionality. Addition of one laser without assigned fluorochrome resulted in decreased fluorescence spill over and improved discrimination of cell subsets. It also increased the staining index when autofluorescence was integrated in the matrix. We conclude that spectral flow cytometry is a highly valuable tool for high-end immunophenotyping, and that fine-tuning of major experimental steps is key for taking advantage of its full capacity.</p

DataSheet_2_Beyond 40 fluorescent probes for deep phenotyping of blood mononuclear cells, using spectral technology.pdf

The analytical capability of flow cytometry is crucial for differentiating the growing number of cell subsets found in human blood. This is important for accurate immunophenotyping of patients with few cells and a large number of parameters to monitor. Here, we present a 43-parameter panel to analyze peripheral blood mononuclear cells from healthy individuals using 41 fluorescence-labelled monoclonal antibodies, an autofluorescent channel, and a viability dye. We demonstrate minimal population distortions that lead to optimized population identification and reproducible results. We have applied an advanced approach in panel design, in selection of sample acquisition parameters and in data analysis. Appropriate autofluorescence identification and integration in the unmixing matrix, allowed for resolution of unspecific signals and increased dimensionality. Addition of one laser without assigned fluorochrome resulted in decreased fluorescence spill over and improved discrimination of cell subsets. It also increased the staining index when autofluorescence was integrated in the matrix. We conclude that spectral flow cytometry is a highly valuable tool for high-end immunophenotyping, and that fine-tuning of major experimental steps is key for taking advantage of its full capacity.</p

DataSheet_4_Beyond 40 fluorescent probes for deep phenotyping of blood mononuclear cells, using spectral technology.pdf

The analytical capability of flow cytometry is crucial for differentiating the growing number of cell subsets found in human blood. This is important for accurate immunophenotyping of patients with few cells and a large number of parameters to monitor. Here, we present a 43-parameter panel to analyze peripheral blood mononuclear cells from healthy individuals using 41 fluorescence-labelled monoclonal antibodies, an autofluorescent channel, and a viability dye. We demonstrate minimal population distortions that lead to optimized population identification and reproducible results. We have applied an advanced approach in panel design, in selection of sample acquisition parameters and in data analysis. Appropriate autofluorescence identification and integration in the unmixing matrix, allowed for resolution of unspecific signals and increased dimensionality. Addition of one laser without assigned fluorochrome resulted in decreased fluorescence spill over and improved discrimination of cell subsets. It also increased the staining index when autofluorescence was integrated in the matrix. We conclude that spectral flow cytometry is a highly valuable tool for high-end immunophenotyping, and that fine-tuning of major experimental steps is key for taking advantage of its full capacity.</p

<i>In vivo</i> replication sites of rZHΔNSs-RLuc RVFV following inoculation via intradermal or intranasal route.

<p>(A) Representative image of luminescence in the lymph node (white arrowhead) draining the right ear of <i>Ifnar1</i>-deficient mouse at 24 h after intradermal inoculation of 10⁴ PFU rZHΔNSs-RLuc. (B) Luminescence signal in the lungs (white arrowhead) at 48 h after intranasal inoculation of 10⁴ PFU rZHΔNSs-RLuc. Images (A) and (B) are representative of five mice. Photographs were overlaid with false colour representation of bioluminescence intensity, measured in photon/s/cm²/sr and indicated on the scales.</p