Cell Preparation

and

Experimental Lab

Dallas

Cell Lab – Dallas

General Missions of Core C

Production of cells: mouse splenic B cells WEHI cells mouse cardiac myocytes

Ligand Screen: treatments sample preparation

Assays for ligand screen: cAMP intracellular Ca++

Protein traps: cellular expression of baits isolation of protein complexes

Zhen’s picture

The

Cells

111 billion

splenic B cells

A milestone

April 15, 2002 Vol. 1, No. 1 BCAfCS Research Reports www.afcs.org

Purification and Characterization of Mouse Splenic BLymphocytesScientific StaffAlliance for Cellular Signaling LaboratoriesUniversity of Texas Southwestern Medical Center, Dallas, TX and Veteran’s Administration Medical Center, San Francisco, CA

Abstract

Initial efforts of Alliance for Cellular Signaling(AfCS) laboratories have focused on establishing standardand reproducible procedures for purifying B lymphocytes(B cells) from mouse spleen. Our goal was to obtaincultures that permit reliable measurement of short-termsignaling events and changes in gene expression. Thisreport describes a negative-selection procedure for B cellenrichment and characterization of the isolated cellpopulation using multiparameter fluorescence-activatedcell sorting (FACS) analysis. Results show that we obtainapproximately 47 x 10 6 cells per spleen, of which 96%contain the B220 marker typically expressed on B cells.More than 80% of these cells can be further characterizedas resting, mature B2 cells. Conditions for maintainingviable cells in serum-free medium for short-term culturewere defined. Supplemented Iscove’s Modified Dulbecco’sMedium (SIMDM) was most consistent among severalmedia in maintaining cell viability for 24 hours. B cellsalso showed consistent and reproducible signaling

to manipulate gene expression in these cells based onreasonably complete knowledge of their genome. We alsowish to study relatively normal cells from a mammalianorganism. Based on these criteria one of the cells chosenwas the resting B lymphocyte (B cell) of the mouse.By secreting antigen-specific immunoglobulinantibody, the B cell plays an essential role in host defense.Resting B cells reside in the circulation and migrate to thespleen and lymph nodes. Activation of the B-cell receptorby antigen in conjunction with appropriate co-stimulationcauses the resting B cell to proliferate and differentiate intoa plasma cell, which makes and secretes large amounts ofimmunoglobulin antibody. In addition to the antigenreceptor, B cells exhibit a large number of additional cellsurface receptors, which are activated by T-cell surfacemolecules, cytokines, bacterial endotoxin, and otherligands. Immunologists have begun to define key steps insignaling pathways downstream of these receptors thatregulate steps in development of the mature B cell andmodulate responses to antigen (1,2,3).

Ligand Screen – B cells

Cell Lab in Dallas

Produces Cells

Treats Cellswith Ligands

Molecular Biology Lab

Microarray analysis

Protein Lab

P-proteins

Antibody Lab

P-proteins

Pilot project - Cornell

Lipid analysiscAMP

Calcium

Current Requirements

6 Outputs

Calcium

cAMP

RNA (microarray)

P-protein blots

2D gels

Lipids

B cells/assay (millions)

~1.5

1.5

18

18

18

6

Assays/year/6*

11,000

11,000

1000

1000

1000

3000

* Based on 100 billion B cells/year

Ligand Screen

Where are we?

Survey of the ligands

Ca2+ cAMP Immunoblot Conc Ligand

Yes NRAKT P90RSK ERK1

ERK250 mg/ml antigen/anti-Ig

NR NR AKT Stat6 6 ng/ml IL-4

NR NRAKT P90RSK ERK1

ERK210 mg/ml CD40L/CD154

YesMaybe (inhib?)

AKT P90RSK ERK1 ERK2

50 ng/ml SDF1/CXCL12

NR Yes NR 10 mM terbutaline

Yes NRP90RSK ERK1

ERK2200 ng/ml BLC/BCA-1/CXCL13

Yes NR AKT ERK1(?) TBD* SLC (CCL21; 6Ckine)

NR NR NR TBD IL-10NR NR NR TBD BAFF/BlyS (TNF family)

NRMaybe (inhib?)

NR TBD TRAIL

NR NR NR TBD IL-2NR NR AKT TBD DAMGO (?)NR NR NR TBD Dynorphin ANR NR TBD DADLE (?)NR NR NR TBD IL-5NR NR AKT P90RSK TBD IFNgNR NR AKT? TBD TNFaNR NR AKT? TBD IGF-1NR NR ERK1? TBD FasL*

TBD palmitoyl-lysoPCNR NR NR TBD Bombesin

NR Yes NR TBD 2-methyl-thio-ATP

NR NR NR TBD PAFNR NR NR TBD Carbachol

Table 1. Responses of Splenic B Lymphocytes to LigandsFacs Priority

Ly77 CD25 CD44 CD69 CD86

A

CD23 CD69 CD80 CD86

A

Ly77 CD18 CD23 CD25 CD44 CD54 CD69

CD86A

CD18(?) CD23 A

NR A

NR A

NR A

CD86? ACD23? A

NR A

NR BNR BNR BNR BNR B

MHC II? BNR B

Ly77? BNR B

BCD29? C

CD80 CD86 CD44 CD54 CD23

C

CCD80 CD44? C

Experiment DescriptionM7MH01112103.primary_lymphocytes_X00

Abs Call1

M7MH01112104.primary_lymphocytes_Y00

Abs Call2

Mean

AFFX-MURINE_B2_at K00131 mouse b2 repeat sequence from clone mm61 Control Sequence24282 P 22503 P 23393101054_at Ia-associated invariant chain 23426 P 22769 P 2309893086_at immunoglobulin kappa chain variable 28 (V28) 19592 P 17586 P 18589AFFX-b-ActinMur/M12481_3_at M12481 Mouse cytoplasmic beta-actin mRNA (_5 _M _3 represent transcript regions 5 prime Middle and 3 prime respectively) Control Sequence18931 P 17626 P 18279102156_f_at M80423:Mus castaneus IgK chain gene, C-region, 3 end /cds=(0,322) /gb=M80423 /gi=196865 /ug=Mm.46804 /len=323 mRNA18338 P 17575 P 17957102599_at translationally regulated transcript (21 kDa) 18039 P 17025 P 17532AFFX-b-ActinMur/M12481_M_atM12481 Mouse cytoplasmic beta-actin mRNA (_5 _M _3 represent transcript regions 5 prime Middle and 3 prime respectively) Control Sequence16793 P 15845 P 16319AFFX-b-ActinMur/M12481_5_at M12481 Mouse cytoplasmic beta-actin mRNA (_5 _M _3 represent transcript regions 5 prime Middle and 3 prime respectively) Control Sequence15693 P 15670 P 1568293583_s_at immunoglobulin heavy chain 6 (heavy chain of IgM) 15207 P 15323 P 1526597696_r_at ribosomal protein L7 15598 P 14410 P 1500494766_at eukaryotic translation elongation factor 1 alpha 1 14287 P 14794 P 14541100213_f_at ribosomal protein L41 15039 P 14013 P 1452693584_at immunoglobulin heavy chain 6 (heavy chain of IgM) 13410 P 13267 P 1333996358_at ribosomal protein S23 12592 P 11947 P 12270100780_at ribosomal protein S4, X-linked 12101 P 11279 P 1169093120_f_at histocompatibility 2, K region 10960 P 11581 P 11271100379_f_at AI837905:UI-M-AL0-abq-c-01-0-UI.s1 Mus musculus cDNA, 3 end /clone=UI-M-AL0-abq-c-01-0-UI /clone_end=3 /gb=AI837905 /gi=5472118 /ug=Mm.89082 /len=223 mRNA11920 P 10293 P 1110796300_f_at RIKEN cDNA 3200001M24 gene 11436 P 10342 P 10889101487_f_at Mus musculus adult male kidney cDNA, RIKEN full-length enriched library, clone:0610010O05, full insert sequence10613 P 11088 P 1085195215_f_at ubiquitin C 10853 P 10604 P 1072998119_at ribosomal protein L30 11280 P 10148 P 1071496426_at thymosin, beta 4, X chromosome 11214 P 9873 P 10544100694_at ribosomal protein, large, P1 10718 P 10183 P 1045197647_at ribosomal protein S16 10769 P 9976 P 1037398085_f_at ribosomal protein S28 10841 P 9863 P 1035292866_at butyrophilin-like 2 10251 P 9555 P 9903102126_at ribosomal protein S12 10201 P 9342 P 9772101664_at ribosomal protein S3a 9959 P 9395 P 9677101255_at ubiquitin B 10272 P 8971 P 9622101869_s_at hemoglobin beta chain complex 9118 P 9124 P 9121100686_at ribosomal protein S2 9237 P 8971 P 9104100322_at expressed sequence AI893585 8993 P 8885 P 8939101577_at ribosomal protein S6 9552 P 8213 P 8883101573_f_at ribosomal protein L27a 9156 P 8439 P 879896290_f_at ribosomal protein L21 9010 P 8401 P 8706

Current Ligand List

LigandListB.xls

D:\LigandListB.xls

Priority Ligand Abb.

Receptor (IUPHAR name) Receptor (common name)

Standard ligands

A antigen/anti-Ig AIG BCR

A IL-4 I04 IL-4R

A CD40L/CD154 40L CD40

A SDF1/CXCL12 SDF 2.1.CXC.04.MMU.00.00CXCR4

A terbutaline TER 2.1.ADR.B2.MMU.00.00ß2AR

Ligand screen

A BLC/BCA-1/CXCL13 BLC 2.1.CXC.05.MMU.00.00CXCR5

A SLC (CCL21; 6Ckine) SLC 2.1.CC.07.MMU.00.00CCR7

A IL-10 I10 IL-10R

B IFNg IFG IFNgR

B TNFa TNF TNF-RII (and possibly R1)

B IGF-1 IGF

C 2-methyl-thio-ATP 2MA 2.1.NUCT.02.MMU.00.00 & 2.1.NUCT.05.MMU.00.00P2YR

C PGE2 PGE 2.1.PG.EP1.MMU.00.00 - 2.1.PG.EP4.MMU.00.00EP1R, EP2R, EP3R

C LTB4 (leukotriene B4) LB4 LTB4R

C NGF NGF

C Bombesin BOM

D fMLP FML

D Neurokinin B NEB

D Lysophosphatidic acid LPA Edg family (2,4,7)

D ELC (CCL19; MIP3b) ELC 2.1.CC.07.MMU.00.00CCR7

E Sphingosine-1-PhosphateS1P Edg family (1,3,5,6,8)

E CpG-oligodeoxynucleotideCPG Toll Receptor 9

C LPS LPS Toll Like Receptor-4 (or –2)

C TGF-beta TGF TGF-beta R I/II

E maxadilan MAX VIP1R

E RO-25-1553 R25 VIP2R

E CP55,940 C55 2.1.CBD.02.MMU.00.00Cannabinoid CB2

E CD27** 70L CD70

A BAFF/BlyS (TNF family) BAF U, (TNF-R family) (BMP, TACI)C PAF PAF PAFRD MIP3a M3A 2.1.CC.06.MMU.00.00CCR6

These were tested and showed a functional response.

These have not been offcially tested yet?

These were tested and gave no response but should be kept on list because of known

function(s) related to B cell biology.

Current Ligand List31 approved ligands for the initial B cell single-ligand screen

yellow – tested and gave probable response (24) blue – not yet tested (4) tan – no response in original test but should be there (3)

4 additional ligands to be tried based on expression in GNF arrays.

4 ligands - no response in testing but still under re-evaluation

9 ligands - unresponsive in resting cells – probably due to absence of receptors, which are expressed in response to stimuli (use for ligand pairs?)

5 ligands have been rejected.

21 done in triplicate –

up to 18 more to complete single ligand screen

Patterns of Response

Group Ligand Calcium Cyclic AMP P-Protein Blots Genes Other

Gi S1P ++ 0 E/A/R SomeSDF ++ 0 E/A/R SomeBLC ++ 0 E/A/R SomeELC ++ 0 E/A/R SomeSLC ++ 0 E/A/R Some

Gs Terbutaline 0 ++++ (sust) 0 SomePGE2 0 ++ (sust) 0 Some2-MA (?) 0 + 0 Some

Gq & Gs LPA ++ (fast) ++ 0 Some

BCR Antigen ++++ 0 E/A/R Lots Proliferation

IL4 IL4 0 0 Stats 3 & 6 Lots

CD40 Anti-CD40 0 0 Delayed E/A/R Lots Proliferation

IL10 IL10 0 0 Stat 3 Yes

TNF TNFalpha 0 0 0 Yes

IGF IGF1 0 0 0 Yes

BAFF BAFF 0 0 0 Yes Viability

Assays:

cAMP

Challenges

EIA – competition of Peroxidase-cAMP with acetylated cAMP from samples

No PDE inhibitors

Rapid response

Assay interference

Solution

More sensitive assay – low fmol range switch to protocols using acetylation of cAMP

Effects of Propranolol on cAMP Responses

Time (min)

0 5 10 15 20

PropranololForskolinP+ F

Time (min)

0 5 10 15 20

cAM

P (

fmo

l/1x1

06 c

ells

)

0

50

100

150

200

250

300

PropranololTerbutalineP+ T

Terbutaline: 10 MPropranolol: 1 MForskolin: 100 M

10 M terbutaline 10 M PGE2

0.34 nM IL4 1 M LPA

cAMP – Ligand Interactionsc

AM

P (

fmo

l/mill

ion

ce

lls)

0

50

100

150

200

250

300

350

Co

ntr

ol

3 T

ER

Cont BLC ELC SDF SLC

cAM

P (

% o

f co

ntr

ol)

0

20

40

60

80

100

120

140

160+ 3 M terbutaline

Cont BLC ELC SDF SLC

cA

MP

(%

of

con

tro

l)

0

20

40

60

80

100

120

140

160No stimulation

O

N

O

N

COO-COO- -OOC

-OOCCa2+

O

N

O

N

COO-COO- -OOC

-OOCCa2+

EGTA

BAPTA

Fluo-3

O

N

O

N

-OOC-OOCCOO-

COO-

CH3

O O-O

Cl Cl

Ca2+

Calcium assay

• Load cells with dye

• Incubate

• Measure basal fluorescence

• Add ligand – measure fluorescence over time

• Fmax – use detergent to lyse cells and expose dye to saturating Ca to measure

• Fmin - add EDTA to chelate Ca++ to measure basal fluorescent of dye

F – FminFree Ca++ = X Kd Fmax - F

Calcium response Value Std dev.

Peak response 129 nM 35 nM

Peak / baseline 2.03 0.35

Plateau response 82 nM 15 nM

Plateau / baseline 1.31 0.07

Time to peak 24 sec 5 sec

0.2 M BLC

1 mM LPA

Calcium response Value Std dev.

Peak response 130 nM 31 nM

Peak / baseline 1.71 0.07

Plateau response 96 nM 26 nM

Plateau / baseline 1.26 0.03

Time to peak 16 sec 2 sec

0

50

100

150

200

250

300

350

-200 -100 0 100 200 300 400 500 600

Time (sec)

[Ca2

+]

(nM

)

control

control

AIG

AIG

PGE,(10)

PGE,(10)

PGE(10)/AIG

PGE(10)/AIG

PGE(100)

PGE(100)

PGE(100)/AIG

PGE(100)/AIG

Interactions ?

0

50

100

150

200

250

300

350

-50 0 50 100 150 200 250 300

Time (sec)

[Ca2

+]

(nM

)

control

control

AIG

AIG

PGE,(10)

PGE,(10)

PGE(10)/AIG

PGE(10)/AIG

PGE(100)

PGE(100)

PGE(100)/AIG

PGE(100)/AIG

0

50

100

150

200

250

300

350

400

450

-50 0 50 100 150 200 250 300

Time (sec)

[Ca

2+]

(nM

)

control

control

AIG

AIG

TER(10uM)

TER(10uM)

TER(10uM)/AIG

TER(10uM)/AIG

TER(100uM)

TER(100uM)

TER(100uM)/AIG

TER(100uM)/AIG

3.8% 55.5% 86.0% 40.4%

cell

num

ber

cell

num

ber

cell

num

ber

cell

num

ber

Fluo-3 (fluorescence intensity)

Intracellular Calcium Response to Anti-IgM (0.3M)

Response of Ca: (0-5 min) stimulation with anti-IgM

Assays with

WEHI cells

0

200

400

600

800

1000

1200

-200 -100 0 100 200 300 400 500 600

Time (sec)

[Ca2+

] (n

M)

0

0

0.01

0.01

0.03

0.03

0.07

0.07

0.13

0.13

0.27

0.27

Calcium Assay in WEHI-231 CellsDose-dependent response to anti-IgM

0

1000

2000

3000

4000

5000

6000

7000

8000

0 5 10 15 20 25

Time (min)

cA

MP

(fm

ol/

1x

106

ce

lls

)

SIMDM

PGE2

TER

cAMP assay in WEHI-231 cells

Protein-interaction traps

2-hybrid in yeast

Pull-downs with exogenous tagged proteins

Isolation of protein complexes formed with tagged proteins expressed in cells

Co-immunoprecipitation of endogenous complexes

CO-precipitation/mass spectrometry approach

• Express FLAG-tagged bait

• Immunoaffinity pulldown

• Gel separation

• Excised band/trypsin digest

• MS/MS

• Database search

Cell Lab

Expression of baits in WEHI cells

Retroviral Infection

General Strategy

• Express tagged-protein driven by 5’-LTR of virus

• Bis-cistronic expression of a cell surface protein or antibiotic resistance gene for selection of cells expressing bait

Mscv-FTM-YFP

Mscv-neo-EGFP

MSCV-FTM-YFP

Quad Events % gated % totalUL 4.0 0.1 0.1UR 23.0 0.4 0.2LL 3758.0 57.8 37.6LR 2722.0 41.8 27.2

MSCV-NEO-EGFP

Quad Events % gated % totalUL 10.0 0.1 0.1UR 16.0 0.2 0.2LL 2199.0 31.7 22.0LR 4721.0 68.0 47.2

Retroviral Expression of Flourescent protein in WEHI

Mscv-neo-EGFP

MSCV-FTM-YFP

Quad Events % gated % totalUL 4.0 0.1 0.1UR 23.0 0.4 0.2LL 3758.0 57.8 37.6LR 2722.0 41.8 27.2

MSCV-NEO-EGFP

Quad Events % gated % totalUL 10.0 0.1 0.1UR 16.0 0.2 0.2LL 2199.0 31.7 22.0LR 4721.0 68.0 47.2

MSCV-FTM-YFP---CD4

How Long Can the Expression Last?

2 days

0

20

40

60

80

100

No Virus 24hr-1 24hr-2 24hr-3

%

8 weeks

0

20

40

60

80

100

No Virus 24hr-1 24hr-2 24hr-3

%

viability

transduced/living

transduced/total

Current state of retroviral infection

• 50-70% infection efficiency (105-106 cfu/ml)• >90% viability• Expression last over 8 wks

• Neomycin selection

Isolation of protein complexes

Pilot experiments with

CMV-3x-Flag-ERK2(from Melanie Cobb)

Transient expression in HEK293 cells

Prelimary Results: Erk2 Associated

Proteins in 293 cells • spectrin, alpha & beta• ATP-dependent RNA helicase A• chromatin-specific transcription

elongatiopn factor• splicing Factor 3b, subunit 1 & 2• ribosomal protein S6 kinase

(Rsk2P90)• methylosome protein 50 (MEP50)• Human kinesin-related motor protein

EG5 • protein methyltransferase• rErk2• kiaa0122 gene product

• Others…

3x-Flag-Erk2

Goals of the next year

Ligand pair interaction in splenic B cells(reduce material required)

Initiate screen in WEHI line

Initiate screen in cardiac myocytes ??

Optimize protein trap protocols - streamline screens

Continue cooperative development of output assays

Lead Scientist

Zhen Yan

Technical Staff

Angela Alexander

Julie Collins

James Davis

Richard Davis

Jody Girouard

Joella Grossoehme

Read Pierce

Jason Polasek

Meghdad Rahdar

Associate Directors

Don Hilgemann

Richard Scheuermann

Scientific staff

Robert Hsueh

Keng-mean Lin

Yan Ni

The People