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OMIP-022: Comprehensive assessment of antigen-specific human T-cell functionality and memory.

Graves AJ, Padilla MG, Hokey DA - Cytometry A (2014)

View Article: PubMed Central - PubMed

Affiliation: Aeras, Rockville, Maryland USA.

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This flow cytometry antibody panel was developed and optimized for the characterization of CD4+ and CD8+ T-cell memory and functional responses in adult and infant cryopreserved peripheral blood mononuclear cells (PBMC) stimulated with peptide pools to various antigens of interest (Table1)... This panel (Table2) began as an incremental enhancement to OMIP-014, and included markers for dump (fixable viability dye, CD14, and CD19), T-cell phenotype (CD3, CD4, and CD8), Th1 cytokines (IFN-γ and TNF), a Th2 cytokine (IL-4), T-cell proliferation cytokine (IL-2), degranulation (CD107a), and activation (CD154)... CCR7 is responsible for homing T-cells to lymph nodes and is expressed on naïve and central memory T-cells (TCM)... CD45RO is the smallest isoform of CD45, and has been shown to be expressed on effector memory (TEM) and TCM The inclusion of both markers allows for T-cells to be categorized as naïve, TCM, TEM, or effector T-cells... Brilliant Violet 785 was chosen for CD45RO based on antibody availability and minimal spectral overlap to other markers... IL-17A is secreted by Th17 T helper cells and acts to recruit innate immune effector cells to the site of inflammation... As such, we initially replaced the MIP-1β from OMIP-014 with and anti-IL-17A antibody... As IL-17A is lowly expressed compared to CD8 (and as PerCP-Cy5.5 is fluorescently brighter), we exchanged the fluorochromes for these two markers to minimize the impact of the high compensation... As an effect of changing CD8 to Ax700, a reduction in CD8+ events [perhaps an escapee phenomenon ] was noted using Ax700 to detect CD8, but was mitigated by staining for CD8 intracellularly... CD154 was maintained on this panel as emerging evidence indicates its role as a specific and sensitive marker in detecting CD4 response, as well as its roles in upregulating antimicrobial peptides and its necessity for T-cell activation of B cells... Furthermore, this panel evolved from an initial desire to implement OMIP-014... Unlike these panels, however, our panel includes T-cell memory markers as well as an extensive combination of cytokines and functions associated with tuberculosis vaccine research.

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Example staining of adult human PBMC following stimulation. (A) The first two rows demonstrate the gating hierarchy from total sample to CD4/CD8 identification. A time gate is used to exclude pressure aberrations from the cytometer that may have occurred during sample acquisition. Aggregate gates are used to exclude brightly positive events that may result from antibody or cell aggregation. The bottom row demonstrates gating for cytokines and functions from CD4+ (top half) and CD8+ (bottom half) events resulting from stimulation with Staphylococcal enterotoxin B. Note that IL-17A and CD154 were gated on the same plot to avoid mischaracterization resulting from the increased spectral overlap observed from their fluorochromes. (B) Example plots showing memory profile of CCR7 versus CD45RO in CD4+ and CD8+ populations. Using CMV pp65 peptide pool-stimulated PBMC from a CMV-reactive donor, IFN-γ+ events (blue) were overlaid onto these plots (gray), confirming localization of these events to the effector memory and effector compartments.
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fig01: Example staining of adult human PBMC following stimulation. (A) The first two rows demonstrate the gating hierarchy from total sample to CD4/CD8 identification. A time gate is used to exclude pressure aberrations from the cytometer that may have occurred during sample acquisition. Aggregate gates are used to exclude brightly positive events that may result from antibody or cell aggregation. The bottom row demonstrates gating for cytokines and functions from CD4+ (top half) and CD8+ (bottom half) events resulting from stimulation with Staphylococcal enterotoxin B. Note that IL-17A and CD154 were gated on the same plot to avoid mischaracterization resulting from the increased spectral overlap observed from their fluorochromes. (B) Example plots showing memory profile of CCR7 versus CD45RO in CD4+ and CD8+ populations. Using CMV pp65 peptide pool-stimulated PBMC from a CMV-reactive donor, IFN-γ+ events (blue) were overlaid onto these plots (gray), confirming localization of these events to the effector memory and effector compartments.

Mentions: CD154 was maintained on this panel as emerging evidence indicates its role as a specific and sensitive marker in detecting CD4 response 27, as well as its roles in upregulating antimicrobial peptides 28 and its necessity for T-cell activation of B cells 29. As indicated in OMIP-014, the inclusion of Brefeldin A in our stimulation protocol requires intracellular staining of CD154. Figure 1 shows an example staining and analysis for adult PBMC stimulated with Staphylococcal enterotoxin B (Fig. 1A) or CMV pp65 (Fig. 1B).


OMIP-022: Comprehensive assessment of antigen-specific human T-cell functionality and memory.

Graves AJ, Padilla MG, Hokey DA - Cytometry A (2014)

Example staining of adult human PBMC following stimulation. (A) The first two rows demonstrate the gating hierarchy from total sample to CD4/CD8 identification. A time gate is used to exclude pressure aberrations from the cytometer that may have occurred during sample acquisition. Aggregate gates are used to exclude brightly positive events that may result from antibody or cell aggregation. The bottom row demonstrates gating for cytokines and functions from CD4+ (top half) and CD8+ (bottom half) events resulting from stimulation with Staphylococcal enterotoxin B. Note that IL-17A and CD154 were gated on the same plot to avoid mischaracterization resulting from the increased spectral overlap observed from their fluorochromes. (B) Example plots showing memory profile of CCR7 versus CD45RO in CD4+ and CD8+ populations. Using CMV pp65 peptide pool-stimulated PBMC from a CMV-reactive donor, IFN-γ+ events (blue) were overlaid onto these plots (gray), confirming localization of these events to the effector memory and effector compartments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231567&req=5

fig01: Example staining of adult human PBMC following stimulation. (A) The first two rows demonstrate the gating hierarchy from total sample to CD4/CD8 identification. A time gate is used to exclude pressure aberrations from the cytometer that may have occurred during sample acquisition. Aggregate gates are used to exclude brightly positive events that may result from antibody or cell aggregation. The bottom row demonstrates gating for cytokines and functions from CD4+ (top half) and CD8+ (bottom half) events resulting from stimulation with Staphylococcal enterotoxin B. Note that IL-17A and CD154 were gated on the same plot to avoid mischaracterization resulting from the increased spectral overlap observed from their fluorochromes. (B) Example plots showing memory profile of CCR7 versus CD45RO in CD4+ and CD8+ populations. Using CMV pp65 peptide pool-stimulated PBMC from a CMV-reactive donor, IFN-γ+ events (blue) were overlaid onto these plots (gray), confirming localization of these events to the effector memory and effector compartments.
Mentions: CD154 was maintained on this panel as emerging evidence indicates its role as a specific and sensitive marker in detecting CD4 response 27, as well as its roles in upregulating antimicrobial peptides 28 and its necessity for T-cell activation of B cells 29. As indicated in OMIP-014, the inclusion of Brefeldin A in our stimulation protocol requires intracellular staining of CD154. Figure 1 shows an example staining and analysis for adult PBMC stimulated with Staphylococcal enterotoxin B (Fig. 1A) or CMV pp65 (Fig. 1B).

View Article: PubMed Central - PubMed

Affiliation: Aeras, Rockville, Maryland USA.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

This flow cytometry antibody panel was developed and optimized for the characterization of CD4+ and CD8+ T-cell memory and functional responses in adult and infant cryopreserved peripheral blood mononuclear cells (PBMC) stimulated with peptide pools to various antigens of interest (Table1)... This panel (Table2) began as an incremental enhancement to OMIP-014, and included markers for dump (fixable viability dye, CD14, and CD19), T-cell phenotype (CD3, CD4, and CD8), Th1 cytokines (IFN-γ and TNF), a Th2 cytokine (IL-4), T-cell proliferation cytokine (IL-2), degranulation (CD107a), and activation (CD154)... CCR7 is responsible for homing T-cells to lymph nodes and is expressed on naïve and central memory T-cells (TCM)... CD45RO is the smallest isoform of CD45, and has been shown to be expressed on effector memory (TEM) and TCM The inclusion of both markers allows for T-cells to be categorized as naïve, TCM, TEM, or effector T-cells... Brilliant Violet 785 was chosen for CD45RO based on antibody availability and minimal spectral overlap to other markers... IL-17A is secreted by Th17 T helper cells and acts to recruit innate immune effector cells to the site of inflammation... As such, we initially replaced the MIP-1β from OMIP-014 with and anti-IL-17A antibody... As IL-17A is lowly expressed compared to CD8 (and as PerCP-Cy5.5 is fluorescently brighter), we exchanged the fluorochromes for these two markers to minimize the impact of the high compensation... As an effect of changing CD8 to Ax700, a reduction in CD8+ events [perhaps an escapee phenomenon ] was noted using Ax700 to detect CD8, but was mitigated by staining for CD8 intracellularly... CD154 was maintained on this panel as emerging evidence indicates its role as a specific and sensitive marker in detecting CD4 response, as well as its roles in upregulating antimicrobial peptides and its necessity for T-cell activation of B cells... Furthermore, this panel evolved from an initial desire to implement OMIP-014... Unlike these panels, however, our panel includes T-cell memory markers as well as an extensive combination of cytokines and functions associated with tuberculosis vaccine research.

Show MeSH