APPLICATION NOTELuminex® Multiplex Immunoassays for Classificationof Th1, Th2, and Th17 CD4+ T Cell Differentiation
Developing CD4+ T cells in the thymus differentiate
into various distinct subsets. Each subset producesa characteristic combination of cytokines to activate
other immune cells, control ongoing immune
responses, or manage immune memory.
Bio-Techne has developed R&D Systems® Luminex®
Immunoassays to specifically quantify biomarkers ofTh1/Th2 and Th9/Th17/Th22 differentiated CD4+T cells. These two Luminex kits allow for thecharacterization of T cell lineages present in CD4+
T cells which have been induced towards specific
T cell subsets.

METHODS
Cell Culture
Using the MagCellectTM Human CD4+T cell Isolation Kit (R&D Systems; Catalog # MAGH102), CD4+T cells
were isolated from Human peripheral blood mononuclear cells cultured in RPMI and supplemented with
10% fetal bovine serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin sulfate.
These cells were either untreated or treated with Th1, Th2 or Th17-inducing reagents.
Th1 Cells: Flasks were coated with 2 µg/mL goat anti-mouse IgG (AF007) overnight at room temperature.
Wash 1X with PBS, and then coat with 1 µg/mL Ms x hCD3ε (MAB100) 2-3 hours at room temperature.
Wash 1X with PBS prior to adding CD4+T cells seeded at 0.5 – 1 x 106/mL. To activate the cells thefollowing polarizing cytokines and antibodies were then added to the flask and incubated for 5 days:
20 ng/ml rhIL-2 (202-IL)
40 ng/ml rhIL-12 (219-IL)

5 µg/ml Ms x hIL-4 (MAB304)
5 µg/ml Ms x hCD28 (MAB342)

A

Th17 cells: Flasks were coated with 2 µg/mL goat anti-mouse IgG (AF007) overnight at room temperature.
Wash 1X with PBS, and then coat with 1 µg/mL Ms x hCD3ε (MAB100) 2-3 hours at room temperature.
Wash 1X with PBS prior to adding CD4+T cells seeded at 0.5 – 1 x 10e6/mL. To activate the CD4+ T cells
the following cytokines and antibodies were then added to the flask and incubated for 5 days:

Concentration (pg/mL)

5 ug/ml PHA

20 ng/ml rhIL-2 (202-IL)

Cell culture supernates were analyzed with either the Luminex Th1/Th2 Fixed Panel (Catalog # LKTM008)
or the Luminex Th9/Th17/Th22 Fixed Panel (Catalog # LKTM009). Samples were assayed according tothe procedures outlined in the product inserts. Both kits are available from R&D Systems.

RESULTS
Cells polarized towards a Th1 phenotype secreted increased levels of IFN-a and TNF-b, as expected for Th1
differentiation (Figure 1B). A hallmark of Th2 differentiation is increased levels of IL-4, IL-5, and IL-13,
which we observed (Figure 1A). Stimulating cells with IL-2, IL-23, IL-1b and IL-6 drove activated CD4+ T celldifferentiation to a Th17 phenotype, as observed by the secretion of IL-17A (Figure 1D).

CONCLUSION
The cytokine profiles measured with our Luminex Fixed Panels for Th1/Th2 and Th9/Th17/Th22 wereconsistent with expected results for each respective T cell phenotype. R&D Systems fixed panels provide aconvenient, efficient, and highly-validated immunoassay option to generate biomarker/cytokine profiles in
response to intracellular and extracellular pathogenic bacteria, viruses, and fungi. These fixed panels areprovided as an off-the-shelf product, making it easier and faster than ever to get theLuminex kits you need.

NT

4000

aCD3/28
Th13000

Th22000

++

10000

+ + +

8000

NT
aCD3/28

6000

Th1+

4000

Th22000+0
IL-12 p70

IL-13

IL-1

IL-4

IL-2

IL-5

GM-CSF

IL-10

IFN-γ

AnalyteCTNF-αD 12000

700
600
500
400
NT300

aCD3/28

200

0

IL-6

Analyte

Th17

IL-10

IL-12p70

IL-13

IL-15 IL-17E

IL-33

IL-4+ +

10000
8000

NT6000aCD3/28+ +4000

Th172000100

Further incubate stimulated cells for 24 hours with PMA (10 ng/mL; Tocris; Catalog # 1201/1) andIonomycin calcium salt (500 ng/mL; Tocris; Catalog # 1704/1)
Luminex Fixed Panel Analysis

5000

Concentration (pg/mL)

Th2 cells: Add CD4+T cells seeded at 0.5 – 1 x 10e6/mL to an un-coated flask. To activate the CD4+ T cells
the following were then added to the flask and incubated for 5 days:

12000

6000

0

Concentration (pg/mL)

Further incubate stimulated cells for 24 hours with PMA (10 ng/mL; Tocris; Catalog # 1201/1) andIonomycin calcium salt (500 ng/mL; Tocris; Catalog # 1704/1)

B 14000

7000

1000

40 ng/mL rhIL-6 (206-IL)
5 µg/mL Ms x hCD28 (MAB342)

20 ng/mL rhIL-2 (202-IL)
20 ng/mL rhIL-23 (1290-IL)
10 ng/mL rhIL-1β (201-LB)

8000

Concentration (pg/mL)

Further incubate stimulated cells for 24 hours with PMA (10 ng/mL; Tocris; Catalog # 1201/1) andIonomycin calcium salt (500 ng/mL; Tocris; Catalog # 1704/1)

0IL-5

CD40 Ligand

IFN-γ

IL-17A

MIP-3αTNF-αAnalyteAnalyteFigure 1 - Th1, Th2 and Th17 Cytokine Differentiation Profiles
A & B - Cytokine profile for the TH1/Th2 fixed panel analytes.
C & D - Cytokine profile for the Th9/Th17/Th22 fixed panel anayltes.
+ = values above the limits of the standard curve• = stimulating cytokine
aCD3/28 = cells from an uncoated flask incubated in RPMI and 10% FBS for 5 days
NT = Non-treated cells from an antibody coated flask with only Ms x hCD28 added for 5 days
Note: The concentrations represented in this figure have not been corrected for the initial 1:2 sample dilution

REFERENCES
1. Suntharalingam, G. et al. (2006)
N. Engl. J. Med. 335:1018.
2. Maude, S.L. et al. (2014) Cancer J. 20:119.
3. Römer, P.S. et al. (2011) Blood 118:6772.
4. Findlay, L. et al. (2010) J. Immunol. Methods
352:1.

5. Stebbings, R. et al. (2013)
J. Immunotoxicol. 10:75.
6. Attarwala, H. (2010). J. Young Pharm. 2:3
7. Hünig, T. et al. (2011) Adv Immunol. 95:111.		
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