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Various Subsets

Intracellular cytokine analysis in polarized Th cell subsets
Intracellular staining of cytokines in cultured Th1, Th2, Th9, Th17, and iTreg cells

Peripheral blood mononuclear cells (PBMCs) were cultured for 14 days in Th1, Th2, Th9, Th17, and iTreg polarization conditions and restimulated with PMA/ionomycin for 5 hours in the presence of BD GolgiStop™ protein transport inhibitor (monensin, Cat. No. 554724). The cells were then fixed with BD Cytofix™ fixation buffer (Cat. No. 554655), permeabilized with BD Perm/Wash™ buffer (Cat. No. 554723), and stained with BD Pharmingen™ antibodies against CD4 and intracellular cytokines IL-9, IFN-γ, IL-4, or IL-17A. The dot plots were derived from a lymphocyte gate. Flow cytometry was performed on a BD™ LSR II system.

Normal human PBMCs were isolated from whole blood by Ficoll-Paque™ Plus, and stimulated and expanded with plate-bound anti-CD3 (10 µg/mL) and soluble anti-human CD28 (1 µg/mL) plus recombinant cytokine IL-2 (10 ng/mL) in the presence of:

  • TH1, IL-12 (50 ng/mL) plus neutralizing mAb to human IL-4 (10 µg/mL)
  • TH2, IL-4 (50 ng/mL) plus neutralizing mAb to IFN-γ (10 µg/mL)
  • TH17, IL-6 (50 ng/mL), IL-1β (10 ng/mL), IL-23 (10 ng/mL), TGF-β (10 ng/mL) plus neutralizing mAb to IFN-γ (10 µg/mL) and IL-4 (10 µg/mL)
  • iTreg, TGF-β (10 ng/mL) plus neutralizing mAb to IFN-γ (10 µg/mL) and IL-4 (10 µg/mL)
  • TH9, IL-4 (50 ng/mL), TGF-β (10 ng/mL), and neutralizing mAb to IFN-γ (10 µg/mL)
Detection of three cytokines simultaneously in CD4+ and CD8+ T cells
Simultaneous detection of TNF, IFN-γ, and IL-2 in CD4+ and CD8+ T cells
Cryopreserved PBMCs were stimulated with SEB (1 µg/mL) in the presence of brefeldin A for 6 hours. Activated cells were lysed and fixed using BD FACS™ lysing solution (Cat. No. 347691), then washed and permeabilized using BD FACS™ Permeabilizing Solution 2 (Cat. No. 340973). Cells were stained with a 6-color antibody panel including BD FastImmune™ cytokine antibodies [TNF FITC (Cat. No. 340511), IFN-γ PE (Cat. No. 340452), IL-2 APC (Cat. No. 341116), CD8 PerCP-Cy™5.5 (Cat. No. 341051), CD4 PE-Cy™7 (Cat. No. 348789), and CD3 BD Horizon™ V450] and analyzed on a BD FACSCanto™ II flow cytometer. Dot plots are based on a lymphocyte (FSC/SSC) and CD3+ gate.
Cytokine expression in naïve, memory, and effector T cells (part A)
Analysis of cytokine expression in CD4+ and CD8+ T-cell subsets using a BD FastImmune protocol
Human PBMCs were isolated from a healthy donor using CPT tubes. Cells were stimulated for 6 hours with SEB (1 µg/mL) in the presence of brefeldin A. Activated cells were then washed and stained with CD3 APC-H7 (Cat. No. 641397), CD4 PerCP-Cy5.5 (Cat. No. 341654), CD8 BD Horizon V500™ (Cat. No. 560774), CD28 APC (Cat. No. 559770), CCR7 PE (Cat. No. 552176), and CD45RA PE-Cy7 (Cat. No. 337167). Cells were then fixed by incubation with BD FACS lysing solution (Cat. No. 347691), washed, and permeabilized using BD FACS Permeabilizing Solution 2 (Cat. No. 340973). Cells were then washed twice, stained with IFN-γ BD Horizon V450 (Cat. No. 560371), IL-2 FITC (Cat. No. 340448), TNF Alexa Fluor® 700 (Cat. No. 557996), and analyzed on a special order BD LSR II cell analyzer. Dot plots are based on a lymphocyte (FSC/SSC), CD3+ and CD4+ gate.
Cytokine expression in naïve, memory, and effector T cells (part B)
Analysis of cytokine expression in naïve, memory, and effector T-cell subsets using a BD FastImmune protocol
Human PBMCs were isolated from a healthy donor using CPT tubes. Cells were stimulated for 6 hours with SEB (1 µg/mL) in the presence of brefeldin A. Activated cells were then washed and stained with CD3 APC-H7 (Cat. No. 641397), CD4 PerCP-Cy5.5 (Cat. No. 341654), CD8 BD Horizon V500™ (Cat. No. 560774), CD28 APC (Cat. No. 559770), CCR7 PE (Cat. No. 552176), and CD45RA PE-Cy7 (Cat. No. 337167). Cells were then fixed by incubation with BD FACS lysing solution (Cat. No. 347691), washed, and permeabilized using BD FACS Permeabilizing Solution 2 (Cat. No. 340973). Cells were then washed twice, stained with IFN-γ BD Horizon V450 (Cat. No. 560371), IL-2 FITC (Cat. No. 340448), and TNF Alexa Fluor® 700 (Cat. No. 557996), and analyzed on a special order BD LSR II cell analyzer. Dot plots are based on a lymphocyte (FSC/SSC), CD3+ and CD4+ gate.

 
Antigen-specific CD4+ IFN-γ responses
Typical CD4 IFN-γ responses to three different antigens measured using the BD FastImmune cytokine system
Heparinized whole blood from seropositive individuals was restimulated with antigen and costimulatory antibodies (CD28 and CD49d) in the presence of the secretion inhibitor brefeldin A, fixed using BD FACS lysing solution (Cat. No. 347691), then washed and permeabilized with BD FACS Permeabilizing Solution 2 (Cat. No. 340973). Samples were analyzed using the BD FastImmune CD4 intracellular IFN-γ detection kit (Cat. No. 340970). Typical frequencies of IFN-γ–producing cells are shown for antigen-restimulated (+Ag) and unstimulated (–Ag) samples.
Transcription factor staining by flow cytometry in two different cell preparations using the BD Pharmingen Transcription Factor Buffer Set
Transcription factor staining by flow cytometry in two different cell preparations using the BD Pharmingen Transcription Factor Buffer Set
A Expression of FoxP3 in human regulatory T cells.
B RORγt and IL-17A expression in BALB/C mouse polarized Th17 cells.

 
 

Th17

Intracellular cytokines (human PBMCs)

Flow cytometric analysis of PE anti-human IL-17A on stimulated PBMCs

Human PBMCs were stimulated with PMA/ionomycin in the presence of BD GolgiStop protein transport inhibitor (Cat. No. 554724) for 5 hours. Cells were then fixed and permeabilized using BD Cytofix/Cytoperm™ reagents (Cat. No. 554714) followed by staining with PE anti-human IL-17A (Cat. No. 560486), and Alexa Fluor® 647 anti-human IFN-γ (Cat. No. 557729; left panel) or APC anti-human IL-4 (Cat. No. 554486; right panel). The dot plots were derived from a CD4+ lymphocyte gate. Flow cytometry was performed using a BD FACSCalibur™ system.

Th17 culture analyzed using the Human Th1/Th2/Th17 Phenotyping Kit
Flow cytometric analysis of a Th17 culture using the Human Th1/Th2/Th17 Phenotyping Kit
PBMCs were stimulated for 13 days with anti-CD3 and anti-CD28 monoclonal antibodies in the presence of recombinant IL-6, TGF-β, IL-1β, and IL-23, as well as anti–IFN-γ and anti–IL-4 neutralizing antibodies. Harvested cells were stained according to the Human Th1/Th2/Th17 Phenotyping Kit (Cat. No. 560751) instructions and analyzed on a BD LSR II flow cytometer. Dot plots are based on a CD4+ lymphocyte gate.
Intracellular IL-17A and IL-17F staining
IL-17A and IL-17F staining using a BD Cytofix/Cytoperm protocol
Human cells polarized toward a Th17 phenotype were characterized for IL-17A and IL-17F expression using the BD Cytofix/Cytoperm protocol. Cells were cultured in the presence of Th17-polarizing cytokines and with a protein transport inhibitor, such as BD GolgiStop (monensin, Cat. No. 554724) or BD GolgiPlug™ (brefeldin A, Cat. No. 555029), to prevent secretion and thus allow accumulation of the cytokine inside the cell. Cells were fixed and permeabilized with BD Cytofix/Cytoperm fixation/permeabilization solution (Cat. No. 554722) to allow fluorescent antibodies to enter the cell and bind to their target cytokines. Cells were then stained with antibodies to CD4, IL-17A (Alexa Fluor® 647-conjugated, Cat. No. 560437), and IL-17F (PE-conjugated, Cat. No. 561198) and then detected by multicolor flow cytometry.
Intracellular cytokines in Th17 cells (time course)
Representative data from a time course of Th17-polarized cells comparing levels of IL-17A with CD4, IFN-γ, and IL-4.
Human PBMCs were stimulated for 14 days with anti-CD3 and anti-CD28 monoclonal antibodies in the presence of recombinant IL-6, TGF-β, IL-1β, and IL-23, as well as anti–IFN-γ and anti–IL-4 neutralizing antibodies. Cells were treated with BD GolgiStop (monensin) inhibitor, fixed and permeabilized with BD Cytofix/Cytoperm buffer, and then stained with BD Pharmingen antibodies against the indicated cytokines. At 6 days there were significant numbers of cells expressing IL-17A, with numbers of cells increasing at day 10 and then leveling off.

 
Multiplexed quantitation of cytokines in Th17 cultures

Measurement of Th17 cultures using the BD CBA Th1/Th2/Th17 Kits

CD4+ human memory T cells isolated from whole blood were stimulated with plate-bound anti-CD3 and soluble anti-CD28 alone (blue) or in the presence of recombinant IL-23 (orange)(left). Naïve CD4+ splenocytes were stimulated with plate-bound anti-CD3 and soluble anti-CD28 alone (blue) or in the presence of recombinant TGF-β and IL-6 (orange)(right).

RORγt expression on mouse splenocytes cultured under Th17 polarization conditions.
RORγt expression on mouse splenocytes cultured under Th17 polarization conditions.
The histograms show the staining of either isotype control or mouse RORγt PE conjugate on different subsets of polarized Th17 cells based on IL-17A and IFN-γ expression. All the cells were derived from live cell–discriminated events with the forward and side light scatter characteristics of single cells. Flow cytometry was performed on a BD LSRFortessa™ system.
Simultaneous detection of cytokines, transcription factors, and surface markers to characterize mouse Th17 cells
Simultaneous detection of cytokines, transcription factors, and surface markers to characterize mouse Th17 cells

In the experimental results shown, cells were isolated from BALB/c mouse thymus and spleen. Thymocytes or splenocytes were surface stained with fluorescent antibodies specific for CD44, CD62L, CD196, or appropriate immunoglobulin isotype controls. Cells were fixed and permeabilized with the BD Pharmingen transcription factor buffer set and then intracellularly stained with fluorescent antibodies specific for RORγt, Foxp3, IL-17A, and IFN-γ.

In the top panel, the cellular expression of IL-17A is compared with the expression of RORγt, Foxp3 (Treg transcription factor) and IFN-γ (Th1 cytokine). As expected in thymocytes, there were many IL-17A+ RORγt+ double-positive cells, while there was essentially no co-expression of IL-17A with Foxp3 or IFN-γ. Splenocytes expressed very little IL-17A. In the bottom panel, further analysis revealed that IL-17A–producing cells from spleen expressed the surface markers CD44 and CD196 (CCR6) but not CD62L, providing additional information on the phenotype of IL-17A+ cells.



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