Units/ml (ka), off-rates (kd) and overall affinities (KA and KD) are listed. 70 subtype A pos. # 184...

14
Supplementary Fig. 1 Ligation of α4β7 by gp120 induces the phosphorylation of the mitogen activated protein kinase p38. Fresh NK cells were either untreated or treated with AN1 gp120, AN1 L182A,D183A gp120, 92Ug037 trimeric gp120 (each at 50nM), or phytohaemagglutinin for 5 min. p38 phosphorylation, as measured by reactivity of cell lysates with phospho specific p38 mAbs is reported as relative fluorescent units. Error bars represent the standard deviation from three replicate treatments. These results are representative of three independent experiments. 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 Units/ml AN1 AN1 L182A/D183A 93Ug037 Trimer PHA

Transcript of Units/ml (ka), off-rates (kd) and overall affinities (KA and KD) are listed. 70 subtype A pos. # 184...

Page 1: Units/ml (ka), off-rates (kd) and overall affinities (KA and KD) are listed. 70 subtype A pos. # 184 subtype B pos. # 184 subtype C pos. # 184 60 50 40 30 20 10 0 I L V 80 70 60 50

Supplementary Fig. 1 Ligation of α4β7 by gp120 induces the phosphorylation of the mitogen activated protein kinase p38. Fresh NK cells were either untreated or treated with AN1 gp120, AN1 L182A,D183A gp120, 92Ug037 trimeric gp120 (each at 50nM), or phytohaemagglutinin for 5 min. p38 phosphorylation, as measured by reactivity of cell lysates with phospho specific p38 mAbs is reported as relative fluorescent units. Error bars represent the standard deviation from three replicate treatments. These results are representative of three independent experiments.

70.0

60.0

50.0

40.0

30.0

20.0

10.0

0.0–

Uni

ts/m

l

AN1

AN1 L1

82A/D

183A

93Ug0

37 T

rimer

PHA

Page 2: Units/ml (ka), off-rates (kd) and overall affinities (KA and KD) are listed. 70 subtype A pos. # 184 subtype B pos. # 184 subtype C pos. # 184 60 50 40 30 20 10 0 I L V 80 70 60 50

100 101 102 103 1040

20

40

60

80

100

% o

f Max

a

100 101 102 103 1040

20

40

60

80

100

b

100 101 102 103 1040

20

40

60

80

100

c

β7α4 β1

IL2 w/o retinoic acidIL2 + retinoic acid

Supplementary Figure 2. Retinoic acid induces the expression of α4 and β7 on PBMCs. PBMCs were cultured for six days in the presence of OKT3 and IL2, and with retinoic acid where specified. (a) Cells stained with an α4 mAb HP2/1. (b) Cells stained with a β7 mAb FIB504. (c) Cells stained with a β1 mAb P4G11. These results are representative of at least three independent experiments however the time required for increased β7 expression to occur varied from donor to donor. ~10-20% of donor PBMCs responded poorly or not at all to RA treatment.

Page 3: Units/ml (ka), off-rates (kd) and overall affinities (KA and KD) are listed. 70 subtype A pos. # 184 subtype B pos. # 184 subtype C pos. # 184 60 50 40 30 20 10 0 I L V 80 70 60 50

a

b

100 101 102 103 1040

20

40

60

80

100

% o

f Max

gp120-PE

gp120 serum #2 1/100gp120 serum #2 1/400gp120gp120 + HP2/1

100 101 102 103 1040

20

40

60

80

100%

of M

ax

gp120-PE

gp120 serum #1 1/1000gp120 serum #1 1/400gp120gp120 + HP2/1

Supplementary Figure 3. Inhibition of gp120 binding to RA-treated CD8+ T cells by HIV+ immune sera. RA-treated CD8 T cells stained with biotinylated AN1 gp120 in the absence or presence of serum from two HIV+ individuals at different dilution, or the α4 mAb HP2/1. (a) Inhibition by serum from a patient with a viral load of 150,000 RNA copies per mL. (b) Inhibition by serum from a patient with a viral load of 40,000 RNA copies per mL.

Page 4: Units/ml (ka), off-rates (kd) and overall affinities (KA and KD) are listed. 70 subtype A pos. # 184 subtype B pos. # 184 subtype C pos. # 184 60 50 40 30 20 10 0 I L V 80 70 60 50

100

80

60

40

20

0100 101 102 103 104

% o

f Max

a b

93Ug037 trimer93Ug037 monomer

FL2-Height93Ug037 trimer + HP2/193Ug037 trimer

80

60

40

20

00.1 0.3 1 3

Competitor concentration (uM)

% In

hibi

tion

c

.5

1.5

2.5

4.5

p24

(pg/

ml)(

10-4

)

3.5

Supplementary Figure 4. Trimeric envelope and virions bind to α4β7 on T cells. (a) Activated CD8+ T cells cultured in the presence of RA stained directly with 93Ug037 trimer in the absence or presence of the α4 mAb HP2/1. (b) Activated CD8+ T cells cultured in the presence of RA stained with biotinylated 93Ug037 trimer (0.1mM) in the presence of increasing concentrations of unlabeled monomeric or trimeric 93Ug037 gp120. gp120 binding is expressed as % inhibition relative to binding in the absence of unlabeled gp120. (c) p24 Gag antigen ELISA of lysates from RA-treated PBMCs incubated at 4°C with HIV-1 Bal in the presence of Mn++, with the addition of Leu3A, HP2/1, MadCAM-Ig, VCAM-1-Ig and E-cadherin-Ig where specified. Error bars represent the standard deviation from the mean of replicate samples. Significance values (two tailed paired t-test) relative to Bal + Leu3a are: Bal + Leu3A + HP2/1 P=0.03; Bal + Leu3A + VCAM-1-Ig P=0.02; Bal + Leu3A + MadCAM-Ig P= 0.02.

Bal

Bal +

Leu3

a

Bal +

HP2/1

Bal +

leu3a

+ H

P2/1

Bal +

VCAM-1

-Ig

Bal +

Leu3

a +

VCAM-1

-lg

Bal +

Leu3

a +

Mad

CAM-lg

Bal +

E-cad

herin

-Ig

Page 5: Units/ml (ka), off-rates (kd) and overall affinities (KA and KD) are listed. 70 subtype A pos. # 184 subtype B pos. # 184 subtype C pos. # 184 60 50 40 30 20 10 0 I L V 80 70 60 50

100

80

60

40

20

0100 101 102 103 104

% o

f Max

100

80

60

40

20

0100 101 102 103 104

% o

f Max

P5.13-59 + Leu3AP5.13-53 + Leu3AP5.13-59 + Leu3A, HP2/1

93MW959 (C) + Leu3A92Ug21-9 (D) + Leu3A93MW959 (C) + Leu3A, HP2/1

a b

Supplementary Figure 5. Variation in α4β7 binding activity among gp120s. a. Comparison of 93MW959 and 92Ug21-9 gp120s binding to RA cultured PBMCs in the presence of Leu3A. Also included is 93MW959 binding in the presence of Leu3A + HP2/1. b. Comparison of P5.13-59 and P5.13-53 gp120s, two subtype B gp120s cloned from the PBMCs of a patient with chronic HIV infection, binding to RA cultured PBMCs in the presence of Leu3A. Also included is P5.13-59 binding in the presence of Leu3A + HP2/1.

Page 6: Units/ml (ka), off-rates (kd) and overall affinities (KA and KD) are listed. 70 subtype A pos. # 184 subtype B pos. # 184 subtype C pos. # 184 60 50 40 30 20 10 0 I L V 80 70 60 50

α 4β7

α 4β1 α 4

moc

km

ock

Mea

n flu

ores

cenc

e in

tens

ity

93U

g037

trim

er

93U

g037

trim

er

93U

g037

trim

er

93U

g037

trim

er

Neu

trav

idin

0

20

40

60

80

100100

80

60

40

20

0100 101 102 103 104

% o

f Max

FL2-Height

α4β7α4β7

100

80

60

40

20

0100 101 102 103 104

% o

f Max

FL2-Height

α4β1

100

80

60

40

20

0100 101 102 103 104

% o

f Max

100

80

60

40

20

0100 101 102 103 104

% o

f Max

100

80

60

40

20

0100 101 102 103 104

% o

f Max

100

80

60

40

20

0100 101 102 103 104

% o

f Max

92Ug21-9 gp120 P5. 13-53 gp120

93MW959 93Ug037trimer

+ Leu3A+ Leu3A + MadCAM-Ig+ Leu3A + VCAM-lg+ Leu3A + HP2/1

FL2-Height FL2-Height

a b

c d

e

Supplementary Figure 6. gp120 binding to α4 integrin and α4β1. (a) Binding of 92Ug21-9 gp120 to RA cultured PBMCs in the presence of Leu3A and Leu3A + MadCAM-Ig (MadCAM-Ig only binds α4β7). (b) Binding of P5.13-53 gp120 to RA cultured PBMCs in the presence of Leu3A and Leu3A + MadCAM-Ig. (c) Binding of 93MW959 gp120 to RA cultured PBMCs in the presence of Leu3A, Leu3A + MadCAM-Ig, and Leu3A + HP2/1. (d) Binding of 93Ug037 trimer to RA cultured PBMCs in the presence of Leu3A, Leu3A + MadCAM-Ig, Leu3A + VCAM-Ig (VCAM-Ig binds both α4β7 and α4β1), and Leu3A + HP2/1. (e) α4β7 and α4β1 transfected 293T cells stained with α4β7, and α1 mAbs (insets) and α4β7, α4β1, and α4 transfected 293T cells stained with a 93Ug037 trimer. These results are representative of three or more independent experiments.

α4β1

Page 7: Units/ml (ka), off-rates (kd) and overall affinities (KA and KD) are listed. 70 subtype A pos. # 184 subtype B pos. # 184 subtype C pos. # 184 60 50 40 30 20 10 0 I L V 80 70 60 50

ka (1/Ms)kd (1/s)KA (1/M)KD (M)χ2

sCD4 Binding to:

-2

1.7

5.4

9.1

12.8

16.5

20.2

23.9

27.6

31.3

35

0 50 100 150 200 250

AN1 gp120

-3

1

5

9

13

17

21

25AN1L182A,D183A

sCD4 vs:AN1 AN1L182A,D183A

9.31e4 8.65e4

1.32e-3 1.22e-3

7.34e7 7.22e7

1.46e-8 1.40e-8

0.0426 0.0809

b12 Binding to:

-1258

1114172023262932353841444750

AN1 gp120

-1

2.1

5.2

8.3

11.4

14.5

17.6

20.7

23.8

26.9

30

0 50 100 150 200 250 300

AN1L182A,D183A

b12 vs:AN1 AN1L182A,D183A

1.58e6 2.64e6

1.19e-4 2.29e-4

1.33e10 1.15e10

7.5e-11 8.70e-11

0.0409 0.0502

447-52D Binding to:

-2

1.7

5.4

9.1

12.8

16.5

20.2

23.9

27.6

31.3

35AN1 gp120

-2

2

6

10

14

18

22

26

30

0 50 100 150 200 250 300 350 400

AN1L182A,D183A

447-52D vs:AN1 AN1L182A,D183A

2.49e5 1.58e5

6.25e-3 8.76e-3

3.98e7 1.80e7

4.37e-8 5.56e-8

0.191 0.132

697-30D Binding to:

-2

4.7

11.4

18.1

24.8

31.5

38.2

44.9

51.6

58.3

65

AN1 gp120

-2

2

6

10

14

18

22

26

30

0 50 150 250 350 450 550

AN1L182A,D183A

697-30D vs:AN1 AN1L182A,D183A

3.02e3 3.83e3

1.14e-4 3.60e-4

2.64e7 1.06e7

3.78e-8 9.40e-8

0.223 0.113

time (sec)

resp

onse

(RU

)

0 50 100 150 200 250 300time (sec)

resp

onse

(RU

)

time (sec)

0 50 150 250 350 450 550time (sec)

resp

onse

(RU

)re

spon

se(R

U)

time (sec)

resp

onse

(RU

)

0 50 100 150 200 250 300 350 400time (sec)

resp

onse

(RU

)

time (sec)

resp

onse

(RU

)

0 50 100 150 200 250time (sec)

resp

onse

(RU

)

Supplementary Figure 7. Comparison of sCD4 and HIV1 gp120 mAbs binding to AN1 gp120 or AN1L182A,D183A. Kinetics of sCD4 (D1D2), and mAbs b12, 667-30D and 447-52D binding to AN1 gp120 and AN1L182A,D183A as measured by surface plasmon resonance spectroscopy. Data were analysed using BiaEvaluation 4.1 using a 1:1 (Langmuir) binding model. On-rates (ka), off-rates (kd) and overall affinities (KA and KD) are listed.

Page 8: Units/ml (ka), off-rates (kd) and overall affinities (KA and KD) are listed. 70 subtype A pos. # 184 subtype B pos. # 184 subtype C pos. # 184 60 50 40 30 20 10 0 I L V 80 70 60 50

subtype A pos. # 184 subtype B pos. # 184 subtype C pos. # 18470

60

50

40

30

20

10

0I L V

80

70

60

50

40

30

20

10

0I L V

80

70

60

50

40

30

20

10

0I L V

Fre

quen

cy (

%)

Supplementary Figure 8. Replacement of the 4 integrin binding motif LDV with LDI in subtype C HIV gp120. The frequency (%) of the three most common residues (Ile, Leu, Val) in HIV-1 subtypes A, B, and C at position 184 of the V2 loop among the 976 sequences listed in the 2006 HIV Sequence Database is presented for subtypes A, B and C.

Page 9: Units/ml (ka), off-rates (kd) and overall affinities (KA and KD) are listed. 70 subtype A pos. # 184 subtype B pos. # 184 subtype C pos. # 184 60 50 40 30 20 10 0 I L V 80 70 60 50

1000

800

600

400

200

0100 101 102 103 104

Time (min)100 101 102 103 104

FS

H-H

: FS

C-H

eigh

t

100 101 102 103 104

40

30

20

10

00 10 20 30 40

28.4% 22.5%

ICAM-lg PE

mock-treated + IL2 (O/N)10’ gp120-tri w/o IL2 (O/N)

a b c d1000

800

600

400

200

0

1000

800

600

400

200

0

Mock-treated w/o IL2 (O/N

5.4%

% p

os. C

D4+

T C

ells

β2 mAbICAM-1-lg

Supplemental Figure 9. ICAM-1-Ig binding to LFA-1 on CD4+ T cells is increased by gp120-treatment. Highly purified CD4+ T cells cultured for >7 days in the presence of IL2/RA stained with biotinylated ICAM-1-Ig, followed by neutravidin-PE. (a) Mock-treated CD4+ T cells, stained with ICAM-1-Ig. (b) Same cells treated with 93Ug037 gp120 trimer for 10 min., then stained with ICAM-1-Ig. (c) Same CD4+ T cells cultured overnight in media with IL2/RA, treated with a mock protein preparation, then stained with ICAM-1-Ig. (d) CD4+ T cells cultured overnight in media without IL2/RA, treated with 93Ug037 trimer for 10-30 min. Staining with a conformation insensitive 2 mAb is included for reference. Dashed line represents staining with neutravidin PE alone.

Page 10: Units/ml (ka), off-rates (kd) and overall affinities (KA and KD) are listed. 70 subtype A pos. # 184 subtype B pos. # 184 subtype C pos. # 184 60 50 40 30 20 10 0 I L V 80 70 60 50

0 200 400 600 800 1000

SSC-H: SSC-Height

0

200

400

600

800

1000

FS

C-H

: FS

C-H

eigh

t

100 101 102 103 1040

20

40

60

80

100%

of M

ax

100 101 102 103 10 40

20

40

60

80

100

100 101 102 103 104100

101

102

103

10460.7 2.01

0.7736.5

36.4

100 101 102 103 104100

101

102

103

10410.9 53.6

341.52

18.5

100 101 102 103 104100

101

102

103

10411.7 49.8

36.12.3

14.3

R1

R2

gp120-treated

R1 R2

MEM-148100 101 102 103 104

0

200

400

600

800

1000F

SC

-H: F

SC

-Hei

ght

2.31

MEM-148

Mock-treated R1 and R2

β7 β7

β7 β7CD45RA

CD

62L

CD

62L

R2 R2 R2w/o IL2 + IL2

w/o IL2

b

100 101 102 103 1040

200

400

600

800

1000

33.2

gp120-treated R1 and R2

c

d e f

gh i

0 200 400 600 800 10000

200

400

600

800

1000

R2

a

R1

SSC-H: SSC-Height

% o

f Max

FS

C-H

: FS

C-H

eigh

t

FS

C-H

: FS

C-H

eigh

tC

D62

L

Supplementary Figure 10. Subset analysis of LFA-1 induced CD4+ T cells. (a) Purified CD4+ T-cells cultured in RA for 7 days, followed by overnight IL2 starvation, were treated with 93Ug037-trimer for 10’ and stained with the 2 mAb MEM148. Light scatter of IL2 starved cells and segregation in to two regions, R1 and R2 based on size and granularity. (b, c) MEM-148 reactivity in mock-treated vs. gp120-trimer-treated cells. (d) MEM-148 reactivity of cells are displayed in purple in the SSC/FSC dot plot, and are found almost exclusively in R2. (e,f) CD4+ T cells can be segregated into β7

hi and β7lo. 7 expression was displayed for both R1 and R2, indicating

that β7hi expression is found almost entirely in R2. Therefore MEM-148-reactivity resides primarily in the β7

hi

population. (g) A display of cells cultured o/n w/o IL2 were further analyzed for memory phenotype using CD45 RA and CD62L. Virtually all cells in R2 were found to be CD45RA- (memory). (h,i) Cells starved of IL2 overnight were compared with cells maintained in IL2. CD62L/7 staining of the R2 populations is displayed. A slight increase in CD62L is observed in the IL2 starved β7

hi cells. Overall the single marker that best predicts LFA1-1 induction by gp120 is a β7

hi phenotype, although only a subset of this population activates LFA-1. These cells exhibit a memory phenotype.

Page 11: Units/ml (ka), off-rates (kd) and overall affinities (KA and KD) are listed. 70 subtype A pos. # 184 subtype B pos. # 184 subtype C pos. # 184 60 50 40 30 20 10 0 I L V 80 70 60 50

Supplementary Methods

Recombinant envelope proteins.

The following gp120s were employed in these studies: 93MW959 (GenBank accession #

U08453, R5-tropic), 92TH14-12 (GenBank accession #U08801, R5-tropic), 93Ug037

(GenBank accession # U51190, R5-tropic), AN1 gp120 1 (sequence available at

http://ubik.mullins.microbiol.washington.edu/HIV/Doria-Rose2005/, R5-tropic),

92Ug21-9 (GenBank accession # U08804, X4-tropic). Two gp120s, P.13-53 (GenBank

accession # AF138157), and P.13-59 (GenBank accession # AF138158), were cloned

from a chronically infected patient as described elsewhere 2. All proteins were produced

and purified in an identical manner. The mature coding sequences of each envelope

protein, from +1 to the gp120-gp41 junction were inserted into a mammalian expression

vector downstream of a synthetic leader sequence. Vectors were transfected into either

293T human fibroblasts or DHFR- CHO cells (ATCC) using either CaPO4 or Polyfect

(Qiagen). For stable cell lines, cultures were selected in nucleoside-free media and

subjected to increasing concentrations of methotrexate (Sigma). Clonal cell lines were

established and subsequently seeded into hollow-fiber cartridges (30 kD MW

cutoff)(Fibercell Systems). Protein containing supernatants were harvested daily from

the extra-capillary space. Pooled supernatants were passed over a galanthus nivalis lectin

column (Vector Labs). gp120 was eluted with 500mM α-methyl-manno pyranoside

(Sigma), desalted and passed through a cobalt-chelating column to remove contaminants.

Protein was then passed over a superdex-200 26/60 gel-filtration column (GE Healthcare

Bio-Sciences), and peak fractions were collected pooled and concentrated with a stirred

cell concentrator (Millipore). Trace endotoxins were removed from purified protein

preparations by triton X114 extraction (Sigma), followed by extensive dialysis in HEPES

buffered saline pH 7.43. Endotoxin removal was verified by Limulus Amoebocyte Lysate

Chromogenic Endpoint Assay (LAL) (Cambrex). Proteins were quantitated by UV

adsorption at O.D λ280 (extinction coefficient 1.1) and values were confirmed by a

bicinchoninic acid protein assay (Pierce). The recombinant AN1 envelope used to

construct a gp120 affinity column was expressed in CHO-lec cells (ATTC). The

AN1L182A,D183A variant of AN1 was constructed by inserting a DraIII-MluI fragment

Page 12: Units/ml (ka), off-rates (kd) and overall affinities (KA and KD) are listed. 70 subtype A pos. # 184 subtype B pos. # 184 subtype C pos. # 184 60 50 40 30 20 10 0 I L V 80 70 60 50

in the wild-type AN1 gp120 expression vector with a synthetic fragment ( DNA 2.0) that

incorporated the L182A and D183A amino acid substitutions. A chimeric SF162P3’ was

constructed by inserting a synthetic DNA fragment corresponding to nucleotides 98-1286

of SF162P3, which encompasses V1 through V5 domains of the gp120 protein, into the

DraIII-MluI sites of an AN1gp120 expression vector. The SF163P3 sequence was codon

optimized to increase protein expression (DNA 2.0). Similarly, a synthetic fragment

corresponding to the same fragment of SF33A2 was cloned into the DraIII-MluI sites of

an AN1 gp120 expression vector to create SF33A2’ gp120.

gp120-induced p38 phosphorylation in NK cells.

Freshly isolated CD4 negative NK cells were obtained by negative selection using

magnetic beads (StemCell Technologies). Cells were cultured in RPMI containing 10%

FBS + 200U/ml IL2 + 10nM RA. Cells were treated with 50nM gp120 or

phytohaemagglutinin (2 µg/ml) for 5 minutes at 37°C. Cells were immediately pelleted

and lysed with Phospho-Safe extraction reagent (Novagen) followed by denaturation with

Flex Set buffer (BD Bioscences). Lysates (10 µg) were reacted with phospho-p38 mAb

conjugated fluoresceine beads according to the manufacturers instructions, and analyzed

on a BD FACS-array.

Virion capture experiments.

PBMCs were cultured in OKT3 and IL2 and RA as described above. After 6-8 days,

culture medium was washed away with sterile HBS buffer with or without divalent

cations and the cells were resuspended at 1 x 106 cells/ml in a 96 well plate. Buffer

without divalent cations included 10mM EDTA. Cells were preincubated with human

IgG along with blocking agents (anti-CD4 and anti α4 mAbs) where specified. Cells

were then incubated with HIV-1 Bal (Applied Biotechnologies) for 30 minutes on ice,

rinsed thoroughly, and lysed. Bound virus was measured by p24 Gag antigen capture

ELISA (Beckman Coulter).

Page 13: Units/ml (ka), off-rates (kd) and overall affinities (KA and KD) are listed. 70 subtype A pos. # 184 subtype B pos. # 184 subtype C pos. # 184 60 50 40 30 20 10 0 I L V 80 70 60 50

Surface plasmon resonance spectroscopy.

The conformational integrity of the AN1 derivative AN1 L182A/D183A gp120 was

evaluated by measuring the binding kinetics of four conformationally sensitive ligands,

sCD4 and mAbs b12, 447-52D, and 697-30D. sCD4 binding maps to the bridging sheet

between the inner and outer domains of gp1204, b12 maps to the outer-domain, close to

the CD4 binding site5, 447-52D maps to the V3 loop6, and 697-30D maps to the V2

loop7, the region within which we introduced the two alanine substitutions. For

comparison these same ligands were reacted with the w.t. AN1 gp120. AN1 and AN1

L182A,D183A gp120s were immobilized to separate flow cells of a Biacore 30000

optical biosensor using amine coupling chemistry (Biacore, Inc.) to a surface density of

approximately 500RU each. Alcohol dehydrogenase was immobilized to a similar

density in a separate flow cell to control for non-specific interactions. Increasing

concentrations of sCD4 (D1D2) or gp120 mAb b12 were passed sequentially over the

immobilized proteins in running buffer (10mM HEPES, pH 7.4, 10mM NaCl, 3mM

EDTA, 0.01% Tween-20, 0.1% soluble carboxymethyl dextran) at a flow rate of 25

µl/min for 2 min. at 25°C, followed by a dissociation phase of at least 2 min. mAb 697-

30D and 447-52D were captured to a surface density of approximately 600RU each by

protein G previously coupled to the sensor surfaces as described above. mAb 2F5 was

bound to similar densities to a separate flow cell to control for non-specific interactions.

Increasing concentrations of AN1 and AN1 L182A,D183A gp120s were then passed over

these surfaces as described above. The data were analyzed using BiaEvaluation 4.1 using

a 1:1 (Langmuir) binding model.

ICAM-Ig binding to LFA-1 on CD4+ T cells.

The capacity of gp120 to induce the activation of LFA-1 was measured by employing

biotinylated ICAM-1-Ig. Recombinant ICAM-1-Ig (R&D Systems) was biotinylated

using E-Z link NHS biotin (Pierce). Purified CD4+ T cells were cultured in RA, IL2 and

OKT3 for ~7 days and then cultured overnight in the absence of IL2 prior to gp120

treatment. Cells were incubated on ice for 20 min and then treated for 10-30 min at 37°C

with a trimeric gp120 and then stained with biotinylated ICAM-1-Ig followed by

neutravidin PE and analyzed by flow cytometry. Cells not starved of IL2 overnight

Page 14: Units/ml (ka), off-rates (kd) and overall affinities (KA and KD) are listed. 70 subtype A pos. # 184 subtype B pos. # 184 subtype C pos. # 184 60 50 40 30 20 10 0 I L V 80 70 60 50

showed no increase in ICAM-1-Ig binding in response to gp120 treatment indicating that

IL2 starvation was required under the culture conditions we employed.

1. Doria-Rose, N.A. et al. Human immunodeficiency virus type 1 subtype B

ancestral envelope protein is functional and elicits neutralizing antibodies in rabbits similar to those elicited by a circulating subtype B envelope. J Virol 79, 11214-24 (2005).

2. Shankarappa, R. et al. Consistent viral evolutionary changes associated with the progression of human immunodeficiency virus type 1 infection. J Virol 73, 10489-502 (1999).

3. Aida, Y. & Pabst, M.J. Removal of endotoxin from protein solutions by phase separation using Triton X-114. J Immunol Methods 132, 191-5 (1990).

4. Kwong, P.D. et al. Structure of an HIV gp120 envelope glycoprotein in complex with the CD4 receptor and a neutralizing human antibody. Nature 393, 648-59 (1998).

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