Multiplex Analysis of Secreted Biomarkers from Stimulated … · 2015-07-09 · Multiplex Analysis...
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Multiplex Analysis of Secreted Biomarkers from Stimulated Natural Killer Cells R. Fuerstenberg, S. Anderson, K. Brumbaugh, J. Schmidt │ R&D Systems Inc., 614 McKinley PL. NE, Minneapolis, MN, 55413 Abstract NK (natural killer) cells form a branch of the innate immune system that destroys compromised host cells, such as tumor cells or virus- infected cells, by recognizing such cells through a “missing self” mechanism. Over the past decade, NK cells have come under greater scrutiny as major regulators of the host’s immune response via the secretion of cytokines, chemokines, and other factors. In addition, studies of the mechanisms of NK cells as antitumor effector cells have advanced our understanding of potential cancer therapies. NK cells have traditionally been identified phenotypically by the detection of cell surface markers using flow cytometric methods. The known ability of NK cells to secrete cytokines and other factors provides an opportunity to identify the secretory profile of NK cells under specific conditions as an alternative to flow cytometry. In this study, human NK cells were enriched from peripheral blood using a negative selection method and cultured overnight either unstimulated or stimulated with IL-12. The cell culture supernates from both conditions were analyzed for 158 secreted biomarkers in large multiplex immunoassays using R&D Systems Luminex ® Screening Assays. The biomarkers that were produced at increased levels following stimulation were analyzed further to establish a secretory profile for IL-12 activated NK cells. Introduction Human Natural Killer (NK) cells were first recognized as a lymphocyte subset in 1975. Mature NK cells represent approximately 15% of the circulating lymphocytes. 1 In humans, mature NK cells are usually defined as CD3 − CD56 + cells and can be further subdivided based on CD56 expression. In general, CD56 dim NK cells include the majority (90%) of peripheral blood NK cells, whereas CD56 bright NK cells are more abundant in secondary lymphoid tissues. 2 NK cells differ from T and B lymphocytes in that they do not show germ-line receptor rearrangement. Their effector functions are ruled by a mixture of activating and inhibitory receptors. These receptors allow the recognition of altered-self at a cellular level, rather than at the molecular level as with T-cell receptors and immunoglobulins. 1 NK cells recognize and destroy malignant and virus infected cells by responding to signals generated by activating and inhibitory receptors on their surfaces. 2 Upon contact with an appropriate target, NK cells release the membrane-disrupting protein perforin and granzyme proteases from secretory granules. 2 A second essential function of NK cells, especially in viral infections, is to release antiviral cytokines such as IFN-g and TNF-a, which act as immunodefensive agents that serve to activate and recruit resident inflammatory and other cells. 2 These NK cell-drived cytokines regulate dendritic cells, T cells, and B cells and regulate TNF-mediated apoptosis of the NK cells and neighboring cells. 2 Thus, cytokine production by NK cells influences both innate and adaptive immune responses. 2 R&D Systems Luminex Screening Assays are large multiplex panels as tools for discovery. Luminex Screening Assay panels provide the highest plex sizes in the industry and offer an ever increasing menu of biomarkers available for screening. They are excellent tools for a wide array of research topics and require only small volumes of sample. Luminex Screening Assays from R&D Systems maximize efficiency, sample use, and data generation by allowing the quantitative analysis of up to 100 biomarkers in a single assay. In this study, we address the measurement of several protein biomarkers by using R&D Systems Human Luminex Screening Assays (Catalog#LXSAH). Human Luminex Screening Assays are designed for the simultaneous measurement of multiple human biomarkers in cell culture supernates, serum, and plasma. Using the available markers, we evaluated measurable levels in stimulated versus unstimulated NK cells, thereby establishing a secretory biomarker profile. Methods Cells NK cells were prepared from PBMCs using R&D Systems MagCellect Human NK cell Isolation Kit (Catalog#MAGH109). NK cells were either left unstimulated or were stimulated for 24 hours with 10 ng/mL recombinant human IL-12 (Catalog#219-IL/CF). Cell culture supernates were collected and evaluated in R&D Systems Human Luminex Screening Assays. Biomarker Secretion Measurements NK cell supernate samples were diluted 1:2 in Diluent RD6-52 prior to assaying. Luminex Screening Assays (R&D Systems Catalog # LXSAH) were configured using the online ordering tool (http://www.rndsystems.com/ordering_tool.aspx) to allow analysis of 158 biomarkers in the fewest possible assays. Four assays were configured to test all 158 analytes in the current menu. The concentration of each biomarker in each cell culture supernate was quantified by comparison to standard curves. Discussion and Conclusion These results illustrate the broad utility of R&D Systems Luminex Screening Assays by quantitatively comparing 158 different protein biomarkers simultaneously in a small volume of cell culture supernate. Some of our observations are as follow: • Increased levels of secreted biomarkers commonly associated with IL-12 stimulation of NK cells were observed (Figure 3). These included the cytokines TNF-a and IFN-g. • An increase in MPO seems, at first, counterintuitive as MPO is often identified as an inhibitor of NK cell cytotoxicity, reducing its anti- tumor functionality. 3 MPO is been shown in Neutrophils to have antimicrobial activity suggesting a similar role for NK cells. 4,5 • Additionally, higher levels of Chemokines such as CXCL10/IP10, CXCL4/PF4, CCL8/MCP-2, and CCL5/RANTES were also observed in stimulated NK cells when compared to the unstimulated control. Consistant with reports, secretion of these chemo-attractants indicates a role of NK cells in the recruitment of adaptive immune cells. 6 • FGF-21, SPARC, MIF, PCSK9, and many others were also elevated in stimulated versus unstimulated NK cell supernates. These and other examples, when taken together provide a comprehensive secretory profile for NK cells and a basis for further inquiry into the function of NK cells as an integral part of the immune system. Figure 1. Principle of the assay. Figure 2. Table of all the analytes tested. Analyte Luminex Bead Capture Antibody Detection Antibody Streptavidin-PE Streptavidin-PE Target Analyte Detection Antibody Biotin Biotin PE PE Luminex Bead Capture Antibody Target Analyte Streptavidin-PE Polystyrene or Magnetic Magnetic Detection Antibody Biotin PE Luminex Bead Capture Antibody Target Analyte LED (621 nm) LED (511 nm) Light Streptavidin-PE Detection Antibody Biotin PE Luminex Bead Capture Antibody Target Analyte Adiponectin Aggrecan Amphiregulin Angiopoietin-2 APP BAFF BCMA BMP-2 BMP-4 BMP-9 CA125 Calbindin D Cardiac Troponin I CCL2/JE/MCP-1 CCL3/MIP-1a CCL4/MIP-1b CCL7/MCP-3 CCL8/MCP-2 CCL13/MCP-4 CCL17/TARC CCL18/PARC CCL20/MIP-3a CCL22/MDC CCL26/Eotaxin-3 CD14 CD27 CD30 CD40 CD163 CHI3-L1 Cripto-1 CRP CXCL1/GROa CXCL10/IP-10 CXCL11/I-TAC CXCL13/BCL/BCA-1 CXCL16 CXCL4/PF4 CXCL8/IL-8 CXCL9/MIG DcR3 Dkk-1 DR3 DR5/TR2 E Selectin EGF ENA-78 ENPP-2/Autotaxin EphA2 Erythropoietin Factor D Fas Fas Ligand FGF basic FGF-21 FLRG Follistatin Galectin 3 G-CSF GDF-15 GM-CSF Growth Hormone HB-EGF HGF ICAM-1 IFN g IFN-g R1 IGFBP-1 IGFBP-3 IL-1 RI IL-1 RII IL-1a IL-1b IL-1ra IL-2 IL-2 Ra IL-4 IL-5 IL-6 IL-6 Ra IL-9 IL-10 IL-12 p70 IL-13 IL-17 IL-17F IL-18 BPa IL-19 IL-22 IL-23 IL-27 IL-28A IL-29 IL-31 IL-33 IL-34 Leptin M-CSF MFG-E8 MICA MICB MIF MMP-1 MMP-12 MMP-13 MMP-2 MMP-3 MMP-7 MMP-8 MMP-9 Myeloperoxidase Myoglobin NRG1-b1/HRG1-b1 Oncostatin M PCSK9 Pentraxin 3/TSG-14 Periostin/OSF-2 Pro-BNP Progranulin Protein C P-Selectin RAGE RANK L/TRANCE RANTES Renin Resistin ROBO4 SCF Serpin C1 Serpin E1/PAI-1 SHBG SOST SPARC SP-D TACI Tenascin-C TFPI TfR Thrombopoietin Thrombospondin-2 Tie-1 Tie-2 TNF RI TNF RII TNFa Trail R3 ULBP-1 ULBP-2/5/6 ULBP-3 ULBP-4 uPAR Uteroglobin VAP-1 VCAM-1 VEGF VEGF C VEGF R3 vWF STEP 1. Figure 3. A. Heat map showing all the tested biomarkers. Colors correspond to high, mid, and low abundance. B. Quantitative comparison of biomarker concentrations between stimulated and unstimulated NK cells. *The increased IL-12 levels in stimulated cells was due to addition of IL-12 during the stimulation procedure. CXCL8/IL-8 MIF CCL3/MIP-1α CCL4/MIP-1β CXCL4/PF4 IL-12 p70 IL-23 CXCL1/GROα CD14 CCL8/MCP-2 TNFα Myeloperoxidase TNF RII IL-6 TfR ULBP-3 IFN γ CCL5/RANTES Protein C P-Selectin CXCL5/ENA-78 G-CSF FGF-21 Chitinase 3-like 1 SPARC MCP-3 Fas Ligand Serpin E1/PAI-1 PCSK9 IL-1ra CXCL8/IL-8 MIF CCL3/MIP-1α CCL4/MIP-1β CXCL4/PF4 IL-12 p70 IL-23 CXCL1/GROα CD14 CCL8/MCP-2 TNFα Myeloperoxidase TNF RII IL-6 TfR ULBP-3 IFN γ CCL5/RANTES Protein C P-Selectin CXCL5/ENA-78 G-CSF FGF-21 Chitinase 3-like 1 SPARC MCP-3 Fas Ligand Serpin E1/PAI-1 PCSK9 IL-1ra MMP-1 Calbindin D CA125 Aggrecan CXCL10/IP-10 Angiopoietin-2 CCL20/MIP-3α IL-22 IL-34 IL-10 Galectin 3 IGFBP-1 CXCL16 Cardiac Troponin I IL-1β IL-1α MMP-1 Calbindin D CA125 Aggrecan CXCL10/IP-10 Angiopoietin-2 CCL20/MIP-3α IL-22 IL-34 IL-10 Galectin 3 IGFBP-1 CXCL16 Cardiac Troponin I IL-1β IL-1α 10 100 1,000 10,000 100,000 Key Concentration in pg/mL A B STEP 2. STEP 3. References: 1. Cheent, K. et al. (2009) J. Imm. 126:449 2. Reefman, E. et al. (2010) J.Imm. 184(9):4852 3. Betten, A. et al. (2001) J. Leukocyte Biology 70(1):65 4. Klebanoff, S. et al. (2013) J. leukocyte Biology 93(2):185 5. Kumar,V. et al. (2010) International Immunopharmacology 10:1325 6. Roda, J. et al. (2006) Cancer Research 66:517
Transcript of Multiplex Analysis of Secreted Biomarkers from Stimulated … · 2015-07-09 · Multiplex Analysis...
Multiplex Analysis of Secreted Biomarkers from Stimulated Natural Killer CellsR. Fuerstenberg, S. Anderson, K. Brumbaugh, J. Schmidt │ R&D Systems Inc., 614 McKinley PL. NE, Minneapolis, MN, 55413
AbstractNK (natural killer) cells form a branch of the innate immune system that destroys compromised host cells, such as tumor cells or virus-infected cells, by recognizing such cells through a “missing self” mechanism. Over the past decade, NK cells have come under greater scrutiny as major regulators of the host’s immune response via the secretion of cytokines, chemokines, and other factors. In addition, studies of the mechanisms of NK cells as antitumor effector cells have advanced our understanding of potential cancer therapies. NK cells have traditionally been identified phenotypically by the detection of cell surface markers using flow cytometric methods. The known ability of NK cells to secrete cytokines and other factors provides an opportunity to identify the secretory profile of NK cells under specific conditions as an alternative to flow cytometry. In this study, human NK cells were enriched from peripheral blood using a negative selection method and cultured overnight either unstimulated or stimulated with IL-12. The cell culture supernates from both conditions were analyzed for 158 secreted biomarkers in large multiplex immunoassays using R&D Systems Luminex® Screening Assays. The biomarkers that were produced at increased levels following stimulation were analyzed further to establish a secretory profile for IL-12 activated NK cells.
IntroductionHuman Natural Killer (NK) cells were first recognized as a lymphocyte subset in 1975. Mature NK cells represent approximately 15% of the circulating lymphocytes.1 In humans, mature NK cells are usually defined as CD3−CD56+ cells and can be further subdivided based on CD56 expression. In general, CD56dim NK cells include the majority (90%) of peripheral blood NK cells, whereas CD56bright NK cells are more abundant in secondary lymphoid tissues.2
NK cells differ from T and B lymphocytes in that they do not show germ-line receptor rearrangement. Their effector functions are ruled by a mixture of activating and inhibitory receptors. These receptors allow the recognition of altered-self at a cellular level, rather than at the molecular level as with T-cell receptors and immunoglobulins.1
NK cells recognize and destroy malignant and virus infected cells by responding to signals generated by activating and inhibitory receptors on their surfaces.2 Upon contact with an appropriate target, NK cells release the membrane-disrupting protein perforin and granzyme proteases from secretory granules.2
A second essential function of NK cells, especially in viral infections, is to release antiviral cytokines such as IFN-g and TNF-a, which act as immunodefensive agents that serve to activate and recruit resident inflammatory and other cells.2 These NK cell-drived cytokines regulate dendritic cells, T cells, and B cells and regulate TNF-mediated apoptosis of the NK cells and neighboring cells.2 Thus, cytokine production by NK cells influences both innate and adaptive immune responses.2
R&D Systems Luminex Screening Assays are large multiplex panels as tools for discovery. Luminex Screening Assay panels provide the highest plex sizes in the industry and offer an ever increasing menu of biomarkers available for screening. They are excellent tools for a wide array of research topics and require only small volumes of sample. Luminex Screening Assays from R&D Systems maximize efficiency, sample use, and data generation by allowing the quantitative analysis of up to 100 biomarkers in a single assay.
In this study, we address the measurement of several protein biomarkers by using R&D Systems Human Luminex Screening Assays (Catalog#LXSAH). Human Luminex Screening Assays are designed for the simultaneous measurement of multiple human biomarkers in cell culture supernates, serum, and plasma. Using the available markers, we evaluated measurable levels in stimulated versus unstimulated NK cells, thereby establishing a secretory biomarker profile.
MethodsCells NK cells were prepared from PBMCs using R&D Systems MagCellect Human NK cell Isolation Kit (Catalog#MAGH109). NK cells were either left unstimulated or were stimulated for 24 hours with 10 ng/mL recombinant human IL-12 (Catalog#219-IL/CF). Cell culture supernates were collected and evaluated in R&D Systems Human Luminex Screening Assays.
Biomarker Secretion Measurements NK cell supernate samples were diluted 1:2 in Diluent RD6-52 prior to assaying. Luminex Screening Assays (R&D Systems Catalog # LXSAH) were configured using the online ordering tool (http://www.rndsystems.com/ordering_tool.aspx) to allow analysis of 158 biomarkers in the fewest possible assays. Four assays were configured to test all 158 analytes in the current menu. The concentration of each biomarker in each cell culture supernate was quantified by comparison to standard curves.
Discussion and Conclusion These results illustrate the broad utility of R&D Systems Luminex Screening Assays by quantitatively comparing 158 different protein biomarkers simultaneously in a small volume of cell culture supernate. Some of our observations are as follow:
• Increased levels of secreted biomarkers commonly associated with IL-12 stimulation of NK cells were observed (Figure 3). These included the cytokines TNF-a and IFN-g.
• An increase in MPO seems, at first, counterintuitive as MPO is often identified as an inhibitor of NK cell cytotoxicity, reducing its anti-tumor functionality.3 MPO is been shown in Neutrophils to have antimicrobial activity suggesting a similar role for NK cells.4,5
• Additionally, higher levels of Chemokines such as CXCL10/IP10, CXCL4/PF4, CCL8/MCP-2, and CCL5/RANTES were also observed in stimulated NK cells when compared to the unstimulated control. Consistant with reports, secretion of these chemo-attractants indicates a role of NK cells in the recruitment of adaptive immune cells.6
• FGF-21, SPARC, MIF, PCSK9, and many others were also elevated in stimulated versus unstimulated NK cell supernates.
These and other examples, when taken together provide a comprehensive secretory profile for NK cells and a basis for further inquiry into the function of NK cells as an integral part of the immune system.
Figure 1. Principle of the assay.
Figure 2. Table of all the analytes tested.
Analyte
Luminex Bead
Capture Antibody
Detection Antibody
Streptavidin-PE Streptavidin-PE
Target AnalyteDetection AntibodyBiotin
Biotin
PE PE
Luminex BeadCapture Antibody
Target Analyte
Streptavidin-PE
Polystyrene or Magnetic Magnetic
Detection AntibodyBiotin
PE
Luminex BeadCapture Antibody
Target Analyte
LED(621 nm)
LED(511 nm)
LightStreptavidin-PE
Detection AntibodyBiotin
PE
Luminex BeadCapture Antibody
Target Analyte
AdiponectinAggrecanAmphiregulinAngiopoietin-2APPBAFFBCMABMP-2BMP-4BMP-9CA125Calbindin DCardiac Troponin ICCL2/JE/MCP-1CCL3/MIP-1aCCL4/MIP-1bCCL7/MCP-3CCL8/MCP-2CCL13/MCP-4CCL17/TARCCCL18/PARCCCL20/MIP-3aCCL22/MDCCCL26/Eotaxin-3CD14CD27CD30
CD40CD163CHI3-L1Cripto-1CRPCXCL1/GROaCXCL10/IP-10CXCL11/I-TACCXCL13/BCL/BCA-1CXCL16CXCL4/PF4CXCL8/IL-8CXCL9/MIGDcR3Dkk-1DR3DR5/TR2E SelectinEGFENA-78ENPP-2/AutotaxinEphA2ErythropoietinFactor DFasFas LigandFGF basic
FGF-21FLRGFollistatinGalectin 3G-CSFGDF-15GM-CSFGrowth HormoneHB-EGFHGFICAM-1IFN gIFN-g R1IGFBP-1IGFBP-3IL-1 RIIL-1 RIIIL-1aIL-1bIL-1raIL-2IL-2 RaIL-4IL-5IL-6IL-6 RaIL-9
IL-10IL-12 p70IL-13IL-17IL-17FIL-18 BPaIL-19IL-22IL-23IL-27IL-28AIL-29IL-31IL-33IL-34LeptinM-CSFMFG-E8MICAMICBMIFMMP-1MMP-12MMP-13MMP-2MMP-3MMP-7
MMP-8MMP-9MyeloperoxidaseMyoglobinNRG1-b1/HRG1-b1Oncostatin MPCSK9Pentraxin 3/TSG-14Periostin/OSF-2Pro-BNPProgranulinProtein CP-SelectinRAGERANK L/TRANCERANTESReninResistinROBO4SCFSerpin C1Serpin E1/PAI-1SHBGSOSTSPARCSP-DTACI
Tenascin-CTFPITfRThrombopoietinThrombospondin-2Tie-1Tie-2TNF RITNF RIITNFaTrail R3ULBP-1ULBP-2/5/6ULBP-3ULBP-4uPARUteroglobinVAP-1VCAM-1VEGFVEGF CVEGF R3vWF
STEP 1.
Figure 3. A. Heat map showing all the tested biomarkers. Colors correspond to high, mid, and low abundance. B. Quantitative comparison of biomarker concentrations between stimulated and unstimulated NK cells. *The increased IL-12 levels in stimulated cells was due to addition of IL-12 during the stimulation procedure.
CXCL
8/IL
-8
MIF
CCL3
/MIP
-1α
CCL4
/MIP
-1β
CXC
L4/P
F4
IL-1
2 p7
0
IL-2
3
CXCL
1/G
ROα
CD
14
CCL8
/MCP
-2
TN
Fα
Mye
lope
roxi
dase
TN
F RI
I
IL-6
TfR
ULB
P-3
IFN
γ
CCL5
/RAN
TES
Prot
ein
C
P-Se
lect
in
CXCL
5/EN
A-78
G-C
SF
FGF-
21
Chiti
nase
3-li
ke 1
SPAR
C
MCP
-3
Fas
Liga
nd
Serp
in E
1/PA
I-1
PCSK
9
IL-1
ra
CXCL
8/IL
-8
MIF
CCL3
/MIP
-1α
CCL4
/MIP
-1β
CXC
L4/P
F4
IL-1
2 p7
0
IL-2
3
CXCL
1/G
ROα
CD
14
CCL8
/MCP
-2
TN
Fα
Mye
lope
roxi
dase
TN
F RI
I
IL-6
TfR
ULB
P-3
IFN
γ
CCL5
/RAN
TES
Prot
ein
C
P-Se
lect
in
CXCL
5/EN
A-78
G-C
SF
FGF-
21
Chiti
nase
3-li
ke 1
SPAR
C
MCP
-3
Fas
Liga
nd
Serp
in E
1/PA
I-1
PCSK
9
IL-1
ra
MM
P-1
Cal
bind
in D
CA1
25
Agg
reca
n
CXC
L10/
IP-1
0
Ang
iopo
ietin
-2
CCL
20/M
IP-3α
IL-2
2
IL-3
4
IL-1
0
Gal
ectin
3
IGFB
P-1
CXC
L16
Car
diac
Tro
poni
n I
IL-1β
IL-1α
MM
P-1
Cal
bind
in D
CA1
25
Agg
reca
n
CXC
L10/
IP-1
0
Ang
iopo
ietin
-2
CCL
20/M
IP-3α
IL-2
2
IL-3
4
IL-1
0
Gal
ectin
3
IGFB
P-1
CXC
L16
Car
diac
Tro
poni
n I
IL-1β
IL-1α
10100
1,00010,000
100,000
10
100
1,000
10,000
100,000
Key Concentration in pg/mLA
B
STEP 2. STEP 3.
References:1. Cheent, K. et al. (2009) J. Imm. 126:449
2. Reefman, E. et al. (2010) J.Imm. 184(9):4852
3. Betten, A. et al. (2001) J. Leukocyte Biology 70(1):65
4. Klebanoff, S. et al. (2013) J. leukocyte Biology 93(2):185
5. Kumar,V. et al. (2010) International Immunopharmacology 10:1325
6. Roda, J. et al. (2006) Cancer Research 66:517
