US007563584B2
`
`(12) United States Patent
`Perez et al.
`
`(10) Patent No.:
`(45) Date of Patent:
`
`US 7,563,584 B2
`Jul. 21, 2009
`
`(54) METHODS AND COMPOSITIONS FOR
`DETECTING THE ACTIVATION STATE OF
`MULTIPLE PROTEINS IN SINGLE CELLS
`
`(55)
`
`References cited
`U.S. PATENT DOCUMENTS
`
`(75)
`
`Inventors: Omar D. Perez, Stanford, CA (US);
`Garry P. Nolan, San Francisco, CA
`(US)
`
`(73) Assignee: The Board of Trustees of the Leland
`Stanford Junior University, Palo Alto,
`CA (US)
`
`( * ) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 510 days.
`
`(21) Appl.No.: 10/193,462
`
`(22)
`
`Filed:
`
`Jul. 10, 2002
`
`(65)
`
`Prior Publication Data
`
`US 2006/0073474 A1
`
`Apr. 6, 2006
`
`Related U.S. Application Data
`
`(60) Provisional application No. 60/304,434, filed on Jul.
`10, 2001, provisional application No. 60/310,141,
`filed on Aug. 2, 2001.
`
`(51)
`
`Int. Cl.
`(2006.01)
`G01N 33/53
`(52) U.S.Cl.
`......................... .. 435/72,435/2,435/7.21,
`435/723, 435/724, 435/374, 435/375, 435/287.2;
`436/517; 436/540; 436/546; 436/548; 436/10;
`436/35; 436/63; 436/64; 436/172; 422/73,
`422/82.05
`
`(58) Field of Classification Search ................... .. 435/2,
`435/4, 6, 7.1, 7.21, 7.23, 7.24, 7.4, 7.92,
`435/8, 18, 23, 40.5, 375, 973; 436/503, 519,
`436/538, 546, 10, 17, 37, 56, 63, 64, 172,
`436/174; 422/68.1, 82.05, 73
`See application file for complete search history.
`
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`435/7.2
`6,821,740 B2 * 11/2004 Darzynkiewicz et al.
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`6,958,221 B2
`10/2005 Veerapandian et al.
`
`(Continued)
`OTHER PUBLICATIONS
`
`Belloc et a1., Flow Cytometry Detection of Caspase 3 Activation in
`Preapoptotic Leukemic Cells, Cytometry 40: 151-160 (Apr. 2000).
`Date verified by BIOSTIC.*
`
`(Continued)
`
`Primary Examiner—Gail R Gabel
`(74) Attorney, Agent, or Firm—Bozicevic, Field & Francis
`LLP; David C. Scherer
`
`(57)
`
`ABSTRACT
`
`The invention provides methods and compositions for simul-
`taneously detecting the activation state of a plurality of pro-
`teins in single cells using flow cytometry. The invention fur-
`ther provides methods and compositions of screening for
`bioactive agents capable of coordinately modulating the
`activity of a plurality of proteins in single cells. The methods
`and compositions can be used to determine the protein acti-
`vation profile of a cell for predicting or diagnosing a disease
`state, and for monitoring treatment of a disease state.
`
`27 Claims, 101 Drawing Sheets
`
`5 parameter analysis
`phospho-AKTser473
`
`PBMC: IL-1 [3 treated (10 pglml)
`phospho-AKTser473
`88a
`
`phospho-AKTser473
`
`
`
`1
`
`10
`
`100 S 1000
`
`10000
`
`8oo
`
`zwwd-oudsoud
`
`D
`
`1
`
`1000
`100
`10
`phospho-p38
`
`10000
`
`1 I
`
`1
`
`10
`
`1000
`
`10000
`
`
`
`1909
`109
`phospho-p38
`
`19090
`
`Soc:
`
`\-ma-oqdsoqd a2‘ 1
`zwwd-oqdsoud 1
`
`10
`
`100
`
`1000
`
`10000
`
`1000
`100
`10
`phospho-paa
`
`10000
`
`.111 B 411
`phospho-TYK2
`phosphc-TYK2
`phaspho-TYK2
`
`Page 1 of 183
`
`Fluidigm
`Exhibit 1001
`
`Page 1 of 183
`
`Fluidigm
`Exhibit 1001
`
`

`
`US 7,563,584 B2
`Page 2
`
`U.S. PATENT DOCUMENTS
`
`............... .. 436/164
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`6,972,198 B2 * 12/2005 Craig et al.
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`* cited by examiner
`
`Page 3 of 183
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`Fluidigm
`Exhibit 1001
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`Page 3 of 183
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`Fluidigm
`Exhibit 1001
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 1 of 101
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`US 7,563,584 B2
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`Page 4 of 183
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`Fluidigm
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`Page 4 of 183
`
`Fluidigm
`Exhibit 1001
`
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 2 of 101
`
`US 7,563,584 B2
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`
`Page 5 of 183
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`Fluidigm
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`Page 5 of 183
`
`Fluidigm
`Exhibit 1001
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 3 of 101
`
`US 7,563,584 B2
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`Page 6 of 183
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`Fmidigm
`Exhibit 1001
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`Page 6 of 183
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`Fluidigm
`Exhibit 1001
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`

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`U.S. Patent
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`Jul. 21, 2009
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`Sheet 4 of 101
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`US 7,563,584 B2
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`Page 7 of 183
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`U.S. Patent
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`Jul. 21, 2009
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`Sheet 5 of 101
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`US 7,563,584 B2
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`Page 8 of 183
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`Fmidigm
`Exhibit 1001
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`Page 8 of 183
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`Fluidigm
`Exhibit 1001
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`U.S. Patent
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`Jul. 21, 2009
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`Sheet 6 of 101
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`Fluidigm
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`U.S. Patent
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`Jul. 21, 2009
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`Sheet 7 of 101
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`Page 10 of 183
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`Fmidigm
`Exhibit 1001
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`Page 10 of 183
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`Fluidigm
`Exhibit 1001
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`

`
`U.S. Patent
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`Jul. 21, 2009
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`Sheet 8 of 101
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`US 7,563,584 B2
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`Page 11 of 183
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`U.S. Patent
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`Jul. 21, 2009
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`Sheet 9 of 101
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`US 7,563,584 B2
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`Page 13 of 183
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`Fluidigm
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`Page 14 of 183
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`U.S. Patent
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`Jul. 21, 2009
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`Sheet 12 of 101
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`US 7,563,584 B2
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`Fluidigm
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`Page 15 of 183
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`Fluidigm
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`Page 16 of183
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`Fluidigm
`Exhibit 1001
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`Page 16 of 183
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`Fluidigm
`Exhibit 1001
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`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 14 of 101
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`US 7,563,584 B2
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`Fluidigm
`Exhibit 1001
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`Page 17 of 183
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`Fluidigm
`Exhibit 1001
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`Page 18 of 183
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`Fluidigm
`Exhibit 1001
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`U.S. Patent
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`Jul. 21, 2009
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`Sheet 20 of 101
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`US 7,563,584 B2
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`FIG._6B
`
`Page 23 of 183
`
`Fmidigm
`Exhibit 1001
`
`Page 23 of 183
`
`Fluidigm
`Exhibit 1001
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 21 of 101
`
`US 7,563,584 B2
`
`Cytohesin-1
`
`JAB1
`
`Merged
`
`(I)no-92. 2'3
`
`Stimulation
`
`Unstimulated
`
`SICAM-2
`
`oL—JAB1
`
`transfection
`
`CD3 mAb
`
`CD3 mAb
`
`'
`
`' ‘-1
`-
`
`+
`
`1:‘: f"‘1
`_
`+
`_
`
`IP: on-JAB1
`
`1B: on-p-serine
`
`
`
`EPI oz-JAB‘!
`
`[B1 on-JAB1
`
`IP: on-cytohesin-1
`13: 0:-p-threonine
`
`IP: on-cytohesin-1
`1B: o:—cytohesin—1
`
`g_____________V?____?__J
`FIG._6D
`
`Page 24 of 183
`
`Fmidigm
`Exhibit 1001
`
`Page 24 of 183
`
`Fluidigm
`Exhibit 1001
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 22 of 101
`
`US 7,563,584 B2
`
`~-_2<u_m+
`
`~-s_<o_m+uV
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`o_m+
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`
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`
`Relative Cell Number
`
`Page 25 of 183
`
`Fluidigm
`Exhibit 1001
`
`Page 25 of 183
`
`Fluidigm
`Exhibit 1001
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 23 of 101
`
`US 7,563,584 B2
`
`"“°Sp“°'p38
`
`0 CD3
`
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`
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`
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`
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`
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`
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`
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`
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`
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`
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`
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`
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`
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`
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`
`phospho-elk
`
`Minutes
`
`Page 26 of 183
`
`Fluidigm
`Exhibit 1001
`
`Page 26 of 183
`
`Fluidigm
`Exhibit 1001
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 24 of 101
`
`US 7,563,584 B2
`
`c.0om§:m§@_22
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`Page 27 of 183
`
`Fluidigm
`Exhibit 1001
`
`Page 27 of 183
`
`Fluidigm
`Exhibit 1001
`
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 25 of 101
`
`US 7,563,584 B2
`
`m-_>_<o_m\mmn_o\moo
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`
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`
`Page 28 of 183
`
`Fluidigm
`Exhibit 1001
`
`Page 28 of 183
`
`Fluidigm
`Exhibit 1001
`
`

`
`U.S. Patent
`
`02
`
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`
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`
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`
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`
`Page 29 of 183
`
`Fluidigm
`Exhibit 1001
`
`Page 29 of 183
`
`Fluidigm
`Exhibit 1001
`
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 27 of 101
`
`US 7,563,584 B2
`
`
`
`
`
`co:m__._aoa+vn_0umcozcmBoxoom2
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`
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`
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`
`
`Page 30 of 183
`
`Fluidigm
`Exhibit 1001
`
`Page 30 of 183
`
`Fluidigm
`Exhibit 1001
`
`
`
`
`
`
`
`
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 28 of 101
`
`US 7,563,584
`
`B2
`
`CD3+CD4+CD8'CD45RA+CD62L+CD11ad5mCD27+CD28+ gated ceus (as per Figure 3)
`
`Stimuiation:
`1000
`
`CD3/s1CAM-2
`
`SICAM-2
`
`phospho-p44/42
`
`
`FIG._8B
`
`24 hr
`
`48 hr
`
`Stimulus
`
` CD69
`Unstimuiated
`
`CD3/C D28/s1CAM~2
`
`CD3/CD28
`
`SICAM-2
`
`CD28
`
`CD3
`
`Page 31 of 183
`
`Fluidigm
`Exhibit 1001
`
`Page 31 of 183
`
`Fluidigm
`Exhibit 1001
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 29 of 101
`
`US 7,563,584 B2
`
`6 hr mtraceflular IL-2
`
`Intensity
`
`9 10 ”
`E
`‘
`E’
`§ 0.1
`0 0.01
`
`9 10
`%.
`E ‘
`<2:
`0.1 _
`(2%
`0.01
`
`0.001
`
`6111*
`
`19099132
`
`1000
`
`100
`
`CD69
`
`M 0
`
`100
`
`
`1000100
`100
`10
`1000100
`10
`100
`100010000
`
`CD25
`
`FIG._9B
`
`12 hr
`
`10000 Unstimulated
`093101323.
`
`
`
`_
`
` 1
`
`10001000
`
`10K
`
`10
`
`IL-2
`
`FIG._9C
`
`Page 32 of 183
`
`Fmidigm
`Exhibit 1001
`
`
`
`
`1
`
`0.001
`
`0.001 0.01
`
`0.1
`
`1
`
`10
`
`00010.01
`
`01
`
`1
`
`10
`
`CD3 (mg/mi)
`
`CD3{CD28 {(1 :1) mgiml)
`
`' '
`0.001
`0.001
`
`.
`
`1
`
`.
`
`.
`
`s1CAM-2 (mg/mt)
`CD3/CD28
`
`10
`
`(1‘=*}"‘9’m')
`41.4
`
`
`
`Page 32 of 183
`
`Fluidigm
`Exhibit 1001
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 30 of 101
`
`US 7,563,584 B2
`
`51
`
`NFAT-LUC assay
`
`C
`.2
`‘C53
`‘O
`E 2.5-
`E0
`LL
`
`0
`
`CD3
`
`CD3/CD28 CD3/CD28/sICAM-2
`
`10
`
`NF-KB-LUC assay
`
`5
`
`
`
`CD3 CD3/CD28 CD3/CD28/SICAM-2
`
`15
`
`AP-1-LUC assay
`
`CO
`
`C
`.9
`3
`v
`_=.
`
`EOU
`
`.
`
`
`
`FoldInduction
`
`CD3
`
`CD3/CD28 CD3/CD28/slCAM-2
`
`Page 33 of 183
`
`Fmidigm
`Exhibit 1001
`
`FIG 10A
`--
`
`Page 33 of 183
`
`Fluidigm
`Exhibit 1001
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 31 of 101
`
`US 7,563,584 B2
`
`0 3
`'
`
`A655
`
`g_15
`
`CD3/CD28
`CD3/CD28/SICAM-2
`
`O
`
`.
`
`p65
`
`p50
`
`cfos
`
`creb
`
`atf2
`
`cRel
`
`Transcription Factor Nuclear Profile
`
`6
`
`NFAT-LUC assay
`
`CD3/CD28/SICAM-2
`
`
`
`FoldInduction
`
`OD
`
`CD3/CD28
`
`0
`
`5
`
`10
`
`1 5
`
`20
`
`25
`
`Concentration (pg/ml)
`
`FIG._ 100
`
`Page 34 of 183
`
`Fmidigm
`Exhibit 1001
`
`Page 34 of 183
`
`Fluidigm
`Exhibit 1001
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 32 of 101
`
`US 7,563,584 B2
`
`0-3
`
`CD3
`§ CD28
`I s|CAM-2
`
`A655
`
`p65
`
`p50
`
`cFos
`
`creb
`
`aft2
`
`cReI
`
`Transcription Factor Nuclear Profile
`
`FIG._ 10D
`
`Naive CD4+ T cells
`
`180
`
`
`
`FlG._ 10E
`
`Page 35 of 183
`
`Fmidigm
`Exhibit 1001
`
`Page 35 of 183
`
`Fluidigm
`Exhibit 1001
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 33 of 101
`
`US 7,563,584 B2
`
`>-
`
`ZL< 3
`
`"£
`
`TH2>TH1
`
`6hr(intracellular)
`
`Page 36 of 183
`
`Fmidigm
`Exhibit 1001
`
`Page 36 of 183
`
`Fluidigm
`Exhibit 1001
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 34 of 101
`
`US 7,563,584 B2
`
`24 hr (extracellular)
`
`1000
`
`\‘ CD3/CD28/slCAM2
`
`Unstimulated
`
`‘ CD3/CD28
`
`Page 37 of 183
`
`Fmidigm
`Exhibit 1001
`
`Page 37 of 183
`
`Fluidigm
`Exhibit 1001
`
`

`
`U.S. Patent
`
`5,7
`
`2B43
`
`QQ?GE
`
`4..mmWW40.m.3w.s<2_GE
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`
`Page 38 of 183
`
`Fluidigm
`Exhibit 1001
`
`Page 38 of 183
`
`Fluidigm
`Exhibit 1001
`
`
`
`

`
`U.S. Patent
`
`Jul. 21, 2009
`
`Sheet 36 of 101
`
`US 7,