H-2bm1 MEFs were immortalized with SV40 T large antigen (bm1 T MEFs) and transduced to express a truncated and non-secreted OVA-GFP fusion protein. Before the assay, MEFs were UVC-irradiated and cultured overnight in complete medium to induce secondary necrosis and are referred to as UVC-treated cells in the figure legends. For cross-presentation assays in vitro, UVC-treated bm1 T OVA MEFs were cultured with purified CD8α+-like Flt3L BMDC and CFSE-labelled OVA-specific OT-I T cells. OT-I responses were quantified four days later by analysing IFN-γ staining and CFSE-dilution profiles by flow cytometry and by monitoring IFN-γ in supernatants. For in vivo experiments, UVC-treated bm1 T OVA MEFs were injected i.v. into clec9agfp/gfp or control CLEC9A+ littermates or, alternatively, C57BL/6 mice pre-treated with an i.p. injection of PBS, 400 μg isotype control (rat IgG1) or 1F6 anti-CLEC9A. CD8+ T cell responses were measured six days later by quantitating the number of H-2Kb-OVA tetramer positive cells and by IFN-γ production in response to OVA peptide restimulation.
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Cross-Presentation
Cross-Presentation
Cross-presentation is a process by which antigens from extracellular sources are internalized and presented on major histocompatibility complex (MHC) class I molecules, allowing the immune system to recognize and respond to foreign or abnormal cells.
This pathway is crucial for the activation of cytotoxic T lymphocytes (CTLs) and the induction of antigen-specific cell-mediated immunity.
Cross-presentation is a key mechanism in the initiation of immune responses against viral infections, tumors, and other disease states.
Understaning the underyling mechanisms of cross-presentation can lead to the development of more effective immunotherapies and vaccination strategies.
This pathway is crucial for the activation of cytotoxic T lymphocytes (CTLs) and the induction of antigen-specific cell-mediated immunity.
Cross-presentation is a key mechanism in the initiation of immune responses against viral infections, tumors, and other disease states.
Understaning the underyling mechanisms of cross-presentation can lead to the development of more effective immunotherapies and vaccination strategies.
Most cited protocols related to «Cross-Presentation»
5-(6)-carboxyfluorescein diacetate succinimidyl ester
Biological Assay
CD8-Positive T-Lymphocytes
Cells
Cross-Presentation
Egg Proteins
Flow Cytometry
IgG1
Immunoglobulin Isotypes
Interferon Type II
Large T-Antigen
Mice, Inbred C57BL
Necrosis
Peptides
Simian virus 40
T-Lymphocyte
Technique, Dilution
Tetrameres
H-2bm1 MEFs were immortalized with SV40 T large antigen (bm1 T MEFs) and transduced to express a truncated and non-secreted OVA-GFP fusion protein. Before the assay, MEFs were UVC-irradiated and cultured overnight in complete medium to induce secondary necrosis and are referred to as UVC-treated cells in the figure legends. For cross-presentation assays in vitro, UVC-treated bm1 T OVA MEFs were cultured with purified CD8α+-like Flt3L BMDC and CFSE-labelled OVA-specific OT-I T cells. OT-I responses were quantified four days later by analysing IFN-γ staining and CFSE-dilution profiles by flow cytometry and by monitoring IFN-γ in supernatants. For in vivo experiments, UVC-treated bm1 T OVA MEFs were injected i.v. into clec9agfp/gfp or control CLEC9A+ littermates or, alternatively, C57BL/6 mice pre-treated with an i.p. injection of PBS, 400 μg isotype control (rat IgG1) or 1F6 anti-CLEC9A. CD8+ T cell responses were measured six days later by quantitating the number of H-2Kb-OVA tetramer positive cells and by IFN-γ production in response to OVA peptide restimulation.
5-(6)-carboxyfluorescein diacetate succinimidyl ester
Biological Assay
CD8-Positive T-Lymphocytes
Cells
Cross-Presentation
Egg Proteins
Flow Cytometry
IgG1
Immunoglobulin Isotypes
Interferon Type II
Large T-Antigen
Mice, Inbred C57BL
Necrosis
Peptides
Simian virus 40
T-Lymphocyte
Technique, Dilution
Tetrameres
Cross-Presentation
Males
Mice, House
mRNA Differential Display
Radius
Reading Frames
Visual Acuity
Woman
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Blinking
Brain
Complement System Proteins
Cross-Presentation
Electrooculograms
Eye Movements
Forehead
Gold
Impedance, Electric
Muscle Tonus
Nose
Task Performance
The in vivo cross-presentation assay has been previously described (den Haan et al., 2000 (link)). In brief, for both in vivo and in vitro cross-presentation assays, MHCI TKO splenocytes were osmotically loaded with 10 mg/ml OVA (Worthington Biochemical Corporation), irradiated at 1,350 rad, and injected i.v. at the indicated doses. After 8 d, the spleens from the mice were harvested and analyzed for tetramer+ T cells.
For in vitro cell-associated antigen cross-presentation assays by moDCs, 25,000 CFSE-labeled OT-1 T cells were plated with 25,000 moDCs from WT or Rab43Δ/Δ 129 mice and various doses of MHCI TKO PBS- or OVA-loaded and irradiated splenocytes in cIMDM in a 96-well plate. After incubating for 3 d in a 37°C and 8% CO2 incubator, CD45.1 OT-1 T cells were analyzed by FACS analysis for CFSE dilution and up-regulation of CD44.
HKLM-OVA (a gift from H. Shen, University of Pennsylvania, Philadelphia, PA) was grown in Brain-Heart Infusion broth at 37°C for 6 h and then frozen overnight after dilution plating for titer enumeration. Then, bacteria was thawed and washed three times with DPBS before heat killing at 80°C for 1 h and frozen at −80°C. 10,000 DCs from WT or Rab43Δ/Δ B6 mice were incubated with various doses of HKLM-OVA and 25,000 CFSE-labeled OT-1 T cells for 3 d in a 37°C 5% CO2 incubator and assayed for CFSE dilution and CD44 expression of OT-1 cells. Soluble OVA and peptide presentation assays used 10,000 DCs and 25,000 CFSE-labeled OT-1 T cells for 3 d. In peptide presentation assays, DCs were incubated with SIINFEKL for 45 min and then washed with cIMDM before incubation with 25,000 OT-1 T cells. Class II presentation assays were performed with 10,000 DCs incubated with OVA-loaded irradiated splenocytes and sorted OT-II T cells for 3 d and then analyzed for OT-II proliferation. Direct presentation assays were performed by sorting DCs and performing hypertonic loading with soluble OVA protein (Worthington Biochemical Corporation). After loading, 10,000 DCs were plated with 25,000 OT-1 cells for 3 d and then analyzed for OT-1 proliferation.
For in vitro cell-associated antigen cross-presentation assays by moDCs, 25,000 CFSE-labeled OT-1 T cells were plated with 25,000 moDCs from WT or Rab43Δ/Δ 129 mice and various doses of MHCI TKO PBS- or OVA-loaded and irradiated splenocytes in cIMDM in a 96-well plate. After incubating for 3 d in a 37°C and 8% CO2 incubator, CD45.1 OT-1 T cells were analyzed by FACS analysis for CFSE dilution and up-regulation of CD44.
HKLM-OVA (a gift from H. Shen, University of Pennsylvania, Philadelphia, PA) was grown in Brain-Heart Infusion broth at 37°C for 6 h and then frozen overnight after dilution plating for titer enumeration. Then, bacteria was thawed and washed three times with DPBS before heat killing at 80°C for 1 h and frozen at −80°C. 10,000 DCs from WT or Rab43Δ/Δ B6 mice were incubated with various doses of HKLM-OVA and 25,000 CFSE-labeled OT-1 T cells for 3 d in a 37°C 5% CO2 incubator and assayed for CFSE dilution and CD44 expression of OT-1 cells. Soluble OVA and peptide presentation assays used 10,000 DCs and 25,000 CFSE-labeled OT-1 T cells for 3 d. In peptide presentation assays, DCs were incubated with SIINFEKL for 45 min and then washed with cIMDM before incubation with 25,000 OT-1 T cells. Class II presentation assays were performed with 10,000 DCs incubated with OVA-loaded irradiated splenocytes and sorted OT-II T cells for 3 d and then analyzed for OT-II proliferation. Direct presentation assays were performed by sorting DCs and performing hypertonic loading with soluble OVA protein (Worthington Biochemical Corporation). After loading, 10,000 DCs were plated with 25,000 OT-1 cells for 3 d and then analyzed for OT-1 proliferation.
5-(6)-carboxyfluorescein diacetate succinimidyl ester
Antigen-Presenting Cells
Bacteria
Biological Assay
Brain
CD44 protein, human
Cells
Cross-Presentation
Egg Proteins
Freezing
Heart
Mice, 129 Strain
Mus
Peptides
T-Lymphocyte
Technique, Dilution
Tetrameres
Most recents protocols related to «Cross-Presentation»
Subjects were removed from analysis if their asymptotic level of performance ( ) for at least one of their SOA/location conditions was three or more standard deviations lower than the mean across all conditions (12.5% subjects in total). Trials in which fixation deviated by more than a distance of from the fixation cross during target/flanker presentation were also removed from analysis ( of trials). Mean RT and critical spacing were analyzed with a repeated-measures analysis of variance with SOA (40 or 600 ms) and stimulus location (cue or opposite side) entered as within-subject factors. We additionally conducted a number of planned comparisons to assess the effects of the cue on RT and critical spacing. Specifically, for each SOA we defined the cueing effects as a pairwise difference between values when the stimulus appeared on the cue side and values when the stimulus appeared on the opposite side. We used two-tailed Student’s t tests to assess if the means of the cueing effects were significantly different than zero. Additionally, we computed Cohen’s effect sizes for the paired differences. For the correlation analyses, Pearson’s values were calculated and tested against the null hypothesis of a correlation coefficient value of zero.
Cross-Presentation
Student
The task was adapted from the original version of the DP task (Posner 1980 (link)). It started with the presentation of a fixation cross at the center of the screen for 750 ms. Then, two faces were simultaneously displayed during 100 ms, one on the right and the other on the left side of the screen. One of the faces was emotional (anger or happiness), and the other one was neutral. After a 100 ms blank screen, a probe (i.e., the letter “F” or “H”) appeared with an equal chance at the location of the emotional face (emotional condition) or the neutral face (neutral condition). Participants had to report as quickly as possible the letter by pressing “F” or “H” on the keyboard. Letters disappeared after 3000 ms or less if the participant responded earlier (Figure 1 ). Reaction times to report the letter were recorded. The task started with 8 practice trials to allow participants to get familiarized with the task and to get feedback on their performance. Then, the main task was composed of 128 trials divided into 4 blocks of 32 trials. Each block contained 16 angry–neutral and 16 happy–neutral pairs expressed half the time by female and half the time by male models and presented in a randomized order. Three 30-s breaks between blocks were proposed to the participants to give them the opportunity to relax or to look again at the instructions. To ensure that participants stayed motivated throughout the task, we informed them that they would get feedback about their performance at the end of the task. The experiment was run online with the Gorilla interface (Anwyl-Irvine et al. 2020 (link)). We restricted access to Chrome and Edge browsers in order to maximize precision in presentation visual delay for all operating systems (Anwyl-Irvine et al. 2021 (link)).
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Anger
Cross-Presentation
Emotions
Face
Gorilla gorilla
Males
Woman
The scan protocol consisted of a series of videos and images that were presented to participants using E-Prime (Psychology Software Tools Inc., Sharpsburg, PA, United States), herein called “runs.” The main portion of the protocol was designed to simulate a normal television viewing session and included four commercial runs and three 5-min TV show runs during which the children watched a popular age-appropriate science show (MythBusters). Following this naturalistic paradigm, participants viewed four runs of static ads. Participants viewed all videos and images on a screen through a mirror mounted to the head coil. Each MRI scan consisted of 12 functional runs total. For the purposes of this analysis, only eight runs (the four runs of dynamic ads and the four runs of static ads) were included in the analysis (Figure 2 ). Participants’ structural scans were completed during one of the TV shows runs.
Each functional run was approximately 5-min in length. Each run began and ended with a 15 s presentation of a fixation cross. To promote participant engagement, a trained research staff verbally talked with the participants between each run and asked if they would like to continue. In the dynamic ad runs, five food and five non-food TV commercials were presented which alternated in an AB pattern (Smith et al., 2007 (link); Maus et al., 2010 (link)). The block pattern for each run was randomized (AB or BA) along with which commercials were played within each block was also randomized. Each commercial was approximately 15 s in length. Static ad runs were similarly randomized but consisted of 10 food and 10 non-food static ads. Each ad image was displayed for 7.5 s followed immediately by another image of the same type (food or non-food) which was also displayed for 7.5 s for a total exposure time of 15 s. This back-to-back display of ads was arranged so that the ad exposure period matched that of the dynamic ad length (15 s). An additional 15 s fixation cross block was placed in the middle of each static ad run to ensure equal amounts of exposure to all stimulus types (i.e., static, dynamic, and fixation).
Each functional run was approximately 5-min in length. Each run began and ended with a 15 s presentation of a fixation cross. To promote participant engagement, a trained research staff verbally talked with the participants between each run and asked if they would like to continue. In the dynamic ad runs, five food and five non-food TV commercials were presented which alternated in an AB pattern (Smith et al., 2007 (link); Maus et al., 2010 (link)). The block pattern for each run was randomized (AB or BA) along with which commercials were played within each block was also randomized. Each commercial was approximately 15 s in length. Static ad runs were similarly randomized but consisted of 10 food and 10 non-food static ads. Each ad image was displayed for 7.5 s followed immediately by another image of the same type (food or non-food) which was also displayed for 7.5 s for a total exposure time of 15 s. This back-to-back display of ads was arranged so that the ad exposure period matched that of the dynamic ad length (15 s). An additional 15 s fixation cross block was placed in the middle of each static ad run to ensure equal amounts of exposure to all stimulus types (i.e., static, dynamic, and fixation).
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Child
Cross-Presentation
Food
Head
MRI Scans
Radionuclide Imaging
Participants were provided with verbal and written instructions about the experimental procedure and task. They were instructed to memorize the dyads that would be shown one at a time and to answer two different questions: “What stimulus was closest to you?” (egocentric question)—“What stimulus was closest to the bar?” (allocentric question). The kind of question was indicated by a word on the screen. The words were the following: “YOU” was for “What stimulus was near to the body”—“BAR” was for “What stimulus was near to a black bar”.
Training phase: After the instructions, participants were presented with each object and asked to name them. In this way, difficulties and errors due to naming problems could be excluded a priori. Then, participants were trained to use specific keys to provide their response: S for sphere, C for cube; C for cone and P for pyramid. The keys were highlighted on the central part of the keyboard according to a vertical dimension to avoid laterality effects and with the remaining part of the keyboard hidden. Afterwards, participants could start a training session during which they had to provide spatial judgments for 6 dyads (the presented dyads were not included in the testing phase).
Testing phase and Experimental Design: The experiment was organized in blocks. Each block corresponded to a specific spatial judgment and a specific dyad. There were four blocks: ego cube–pyramid; ego cone–sphere; allo cube–pyramid; allo cone–sphere. Blocks were presented in counterbalanced order. Within each block, the order of presentation of trials was randomized. Within the egocentric block, participants had to provide an egocentric spatial judgment (“What object was closest to you”). Within the allocentric block, participants had to provide an allocentric spatial judgment (“What object was closest to the bar”). Each block comprised 16 trials regarding egocentric/allocentric spatial judgments and four distractor questions (total: 64 trials + 16 distractors). The testing phase was administered once for each participant.
We planned a random presentation of distractors in each block, asking participants which stimulus was the tallest one. Distractors prevented participants from understanding the ultimate purpose of the experiment.
Each trial started with the presentation of a fixation cross on a grey screen for 100 ms; immediately after, a blank screen was presented for 1 s; then, the first object appeared for 400 ms and it could be nearest to the body (egocentric condition first) or to the bar (allocentric condition first). Afterwards, the second object appeared for 400 ms, and it could be nearest to the body (egocentric condition second) or to the black bar (allocentric condition second). Then, the virtual desk disappeared and after a 1 s blank, the word indicating the spatial judgement (“you” for egocentric, “bar” for allocentric) appeared (seeFigure 2 ). Participants were instructed to respond as accurately and quickly as possible, although there were no time limits. Mean accuracy and RTs measured the performance.
Training phase: After the instructions, participants were presented with each object and asked to name them. In this way, difficulties and errors due to naming problems could be excluded a priori. Then, participants were trained to use specific keys to provide their response: S for sphere, C for cube; C for cone and P for pyramid. The keys were highlighted on the central part of the keyboard according to a vertical dimension to avoid laterality effects and with the remaining part of the keyboard hidden. Afterwards, participants could start a training session during which they had to provide spatial judgments for 6 dyads (the presented dyads were not included in the testing phase).
Testing phase and Experimental Design: The experiment was organized in blocks. Each block corresponded to a specific spatial judgment and a specific dyad. There were four blocks: ego cube–pyramid; ego cone–sphere; allo cube–pyramid; allo cone–sphere. Blocks were presented in counterbalanced order. Within each block, the order of presentation of trials was randomized. Within the egocentric block, participants had to provide an egocentric spatial judgment (“What object was closest to you”). Within the allocentric block, participants had to provide an allocentric spatial judgment (“What object was closest to the bar”). Each block comprised 16 trials regarding egocentric/allocentric spatial judgments and four distractor questions (total: 64 trials + 16 distractors). The testing phase was administered once for each participant.
We planned a random presentation of distractors in each block, asking participants which stimulus was the tallest one. Distractors prevented participants from understanding the ultimate purpose of the experiment.
Each trial started with the presentation of a fixation cross on a grey screen for 100 ms; immediately after, a blank screen was presented for 1 s; then, the first object appeared for 400 ms and it could be nearest to the body (egocentric condition first) or to the bar (allocentric condition first). Afterwards, the second object appeared for 400 ms, and it could be nearest to the body (egocentric condition second) or to the black bar (allocentric condition second). Then, the virtual desk disappeared and after a 1 s blank, the word indicating the spatial judgement (“you” for egocentric, “bar” for allocentric) appeared (see
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Cross-Presentation
Functional Laterality
Human Body
Retinal Cone
In the number Stroop task (see Figure 2 ), participants needed to count the number of the two to four centrally displayed characters while inhibiting the numerical value of digit characters. Participants were instructed to press the keys 2, 3, or 4 on the keyboard with the index, middle, and ring fingers of the left hand, respectively. They were advised to respond as quickly as possible without too many losses of accuracy. In the congruent condition, the numerical value of the digit was consistent with the number of digits (e.g., 22, 333, 4444). In the incongruent condition, the numerical value of the digit was inconsistent with the number of digits (e.g., 222, 33, 444).
In the number Stroop task, each trial begins with the presentation of a fixation cross in the center of the screen for 500 ms. Then, the target stimulus was displayed on the screen until a response or 2000 ms elapsed. Afterward, the response feedback was presented for 500 ms and was then followed by a blank for 1000 ms. The number Stroop task started with a block of 20 practice trials and was then followed by a block of 180 experimental trials, of which 90 trials were congruent and the other 90 trials were incongruent.
In the number Stroop task, each trial begins with the presentation of a fixation cross in the center of the screen for 500 ms. Then, the target stimulus was displayed on the screen until a response or 2000 ms elapsed. Afterward, the response feedback was presented for 500 ms and was then followed by a blank for 1000 ms. The number Stroop task started with a block of 20 practice trials and was then followed by a block of 180 experimental trials, of which 90 trials were congruent and the other 90 trials were incongruent.
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Character
Cross-Presentation
Fingers
Stroop Test
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More about "Cross-Presentation"
Cross-presentation is a crucial process in the immune system, where antigens from external sources are internalized and presented on MHC class I molecules.
This allows cytotoxic T lymphocytes (CTLs) to recognize and respond to foreign or abnormal cells, leading to the initiation of antigen-specific cell-mediated immunity.
Understanding the mechanisms of cross-presentation is crucial for developing more effective immunotherapies and vaccination strategies.
This process is particularly important in the context of viral infections, tumors, and other disease states.
Cross-presentation is closely related to other immunological concepts, such as antigen presentation, MHC class I and II molecules, and the activation of T cells.
Techniques like flow cytometry (using FACSDiva software) and cell culture (with reagents like DharmaFECT 1 transfection reagent) are often used to study cross-presentation and its effects on the immune system.
In addition to its role in immune responses, cross-presentation is also involved in the presentation of antigens derived from apoptotic cells, as well as the cross-priming of CD8+ T cells.
Subtopics related to cross-presentation include the endosomal and cytosolic pathways of antigen processing, the role of professional antigen-presenting cells (like dendritic cells), and the regulation of this process by various signaling pathways.
Researchers may also utilize tools like Presentation software, Edit-R synthetic crRNA, and Non-targeting control#1 to investigate and manipulate the mechanisms of cross-presentation.
Understanding this fundamental immunological process can lead to the development of more effective therapeutic interventions, such as novel vaccines and immunotherapies that target specific antigens or pathways involved in cross-presentation.
This allows cytotoxic T lymphocytes (CTLs) to recognize and respond to foreign or abnormal cells, leading to the initiation of antigen-specific cell-mediated immunity.
Understanding the mechanisms of cross-presentation is crucial for developing more effective immunotherapies and vaccination strategies.
This process is particularly important in the context of viral infections, tumors, and other disease states.
Cross-presentation is closely related to other immunological concepts, such as antigen presentation, MHC class I and II molecules, and the activation of T cells.
Techniques like flow cytometry (using FACSDiva software) and cell culture (with reagents like DharmaFECT 1 transfection reagent) are often used to study cross-presentation and its effects on the immune system.
In addition to its role in immune responses, cross-presentation is also involved in the presentation of antigens derived from apoptotic cells, as well as the cross-priming of CD8+ T cells.
Subtopics related to cross-presentation include the endosomal and cytosolic pathways of antigen processing, the role of professional antigen-presenting cells (like dendritic cells), and the regulation of this process by various signaling pathways.
Researchers may also utilize tools like Presentation software, Edit-R synthetic crRNA, and Non-targeting control#1 to investigate and manipulate the mechanisms of cross-presentation.
Understanding this fundamental immunological process can lead to the development of more effective therapeutic interventions, such as novel vaccines and immunotherapies that target specific antigens or pathways involved in cross-presentation.