CTL诱导及杀伤实验

一、抗原肽诱导

CTL

制备

were

separated

from

the

whole

blood

of

HLA-A2+

healthy

controls.

PBMCs (2 x10

6

/ml) were cultured with each of the HLAA* 0201 refolding peptides

at a concentration of 10 uM in RPMI 1640 medium containing 10% FCS and 20 U/ml

recombinant human IL-2 (rhIL-2) in 24-well culture plate. Half of the medium was

changed

at

day

4

with

supplementation

of

rhIL-2

at

20

U/ml.

At

day

7,

cells

were

harvested and tested for the presence of peptide-specific CD8+ T cells by an IFN-

γ

release ELISPOT assay.

---------------

Zhou M, Xu D, Li X, Li H, Shan M, Tang J, Wang M, Wang FS, Zhu X,

Tao H

et al

: Screening and identification of severe acute respiratory

syndrome-associated coronavirus- specific CTL epitopes.

J Immunol

2006,

177(4):2138-2145.

2. PBMCs were separated from heparinized venous blood by Ficoll-Hypaque density

gradient

centrifugation

from

HLAA*0201

normal

donors

and

HCC

patients.

DCs

were

generated

from

peripheral

blood

monocytes

as

described

by

Romani.

Briefly,

PBMCs were seeded into six-well culture plates containing 3 ml RPMI-1640 and 10%

FCS

at5

–

10x10

6

/well.

Plates

were

incubated

in

a

37

o

C

incubator

for

2

h,

then

the

non-adherent

cells

were

removed

and

the

adherent

cells

were

cultured

at

37

o

C

in

RPMI-1640 supplemented with 10% FCS, 1000 U/ml human recombinant GM-CSF

and 500 U/ml human recombinant IL-4. All T cell stimulation was with day 7 DCs.

As described below, DCs were matured with lipopolysaccharide (LPS) on day 6 and

HCA587 antigen was added for effective processing at this time.

Day

6

DCs

were

resuspended

in

RPMI-1640

with

1000

U/ml

GM-CSF,

500

U/ml IL-4 and 10 ng/ml LPS and incubated at 37

o

C. After 24 h cultivation the DCs

were collected, washed and pulsed with 10 m g/ml HCA587 peptide for 3 h at 37

o

C.

The DCs were washed twice for the following stimulation.

CD8+

T

cells

were

isolated

by

positive

selection

with

CD8-Dynal

immunomagnetic beads according to the manufacturer’s instructions.

After washing,

CD8+T cells were co-cultured with HCA587

peptide-loaded DCs in 2 ml RPMI-1640

medium supplemented

with 10% AB serum, recombinant human IL-2

(10 ng/ml) and

IL-6 (500 U/ml). Seven days later, the cultured

T cells were restimulated with freshly

prepared peptide-

pulsed DCs and cultured for another 7 days. After four

consecutive

rounds

of

stimulation,

cultures

were

tested

for

the

presence

of

HCA587-specific

CTLs.

-----------

Li

B,

Wang

Y,

Chen

J,

Wu

H,

Chen

W:

Identification

of

a

new

HLA-A*0201-restricted

CD8+

T

cell

epitope

from

hepatocellular

carcinoma-associated antigen HCA587.

Clin Exp Immunol

2005, 140(2):310-319.

tion of CTLs in PBMCs from healthy donors

：

Dendritic

cells

(DCs)

have

the

unique

capacity

of

activating

naive

T

cells

and

initiating primary T-cell response. Antigen-specific T-cell responses from peripheral

blood

mononuclear

cells

(PBMCs)

can

be

elicited

with

antigenic

peptide-pulsed

autologous

DCs.

We

obtained

PBMCs

from

the

buffy

coat

of

heparinized

whole

blood samples of healthy donors by density gradient centrifugation on the Histopaque

1077. These cells were resuspended in serum-free RPMI 1640 and allowed to adhere

to

six

well

plates

at

a

final

concentration

of

1

x

10

7

cells/3

ml/

well.

After

2

h

of

incubation at 37°

C, non-adherent

cells were gently removed with warm medium by

gently pipetting. The non-adherent cells (effector lymphocytes) were cryopreserved in

FCS supplemented by 10% DMSO. The resultant adherent cells containing DCs were

cultured

in

medium

supplemented

with

800

U/ml

GMCSF

and

1,000

U/ml

IL-4

in

37°

C/5% CO

2

. Every 2 days, one-half of the medium was replaced by fresh medium

containing a double concentration of GM-CSF and IL-4 as indicated above. On day 5,

10

ng/ml

of

recombinant

human

tumor

necrosis

factor

a

(TNF-

a

)

was

added

to

the

medium

to

induce

phenotypic

and

functional

maturation

of

DCs.

After

48

h,

DCs

were pulsed

with

20

ug/ml

peptide in the presence of 3

ug/ml

b2- microglobulin

at

37°

C for 3 h and irradiated at 30 Gy before use. The thawed 2 x10

6

of non-adherent

effector lymphocytes were cocultured with 2 x10

5

peptidepulsed irradiated autologous

DCs in a 24-well plate in the presence of 10 ng/ml recombinant human interleukin-7

(IL-7). After 7 days, lymphocytes were restimulated with peptide- pulsed autologous

PBMCs

in

medium

containing 10 ng/ml

IL-7 and

20 U/ml IL-2. About

20 U/ml of

IL-2

was

added

24

h

later

at

regular

intervals,

2

days

after

each

restimulation.

Lymphocytes were restimulated each week in the same manner. On the seventh day,

after the three rounds of restimulation, cells were harvested and tested by ELISPOT

assay.

--------------

An

altered

peptide

ligand

for

nave

cytotoxic

T

lymphocyte

epitope

of

TRP-2(180

–

188) enhanced immunogenicity.

Cancer Immunol Immunother (2007) 56:319

–

329

第三军医大

吴玉章

tion of CTLs in healthy donors

Dendritic cells are characterized by the unique capacity to activate naive T cells and

initiate

primary

T-cell

response.

Antigen-specific

T-cell

responses

from

peripheral

blood

mononuclear

cells

(PBMCs)

can

be

elicited

with

antigenic

peptide-

or

protein-pulsed autologous DCs. Here, PBMCs were isolated from whole blood of 11

healthy

HLA-A2.1

_

volunteer

donors

by

Ficoll/Hypaque

density

gradient

centrifugation.

Human

peripheral

blood

monocyte-derived

DCs

were

generated

as

previously described by us. On day 5 of culture, 10 ng/mL recombinant human tumor

necrosis

factor

was

added

to

the

medium

to

induce

phenotypic

and

functional

maturation.

Then,

48

hours

later,

DCs

were

pulsed

with

20

ug/mL

peptide

in

the

presence of 3 ug/mL

B2-microglobulin at 37°

C for 3 hours and irradiated at 30 Gy

before

use.

Peripheral

blood

lymphocytes

(PBLs,

2

x10

6

)

were

cocultured

with

2

x

10

5

peptide- pulsed irradiated autologous DCs in a 24-well plate in the presence of 10

ng/mL

recombinant

human

interleukin-7.

The

next

day,

recombinant

human

IL-10

was added to the culture medium, to give a final concentration of 10 ng/mL. After 7

days, lymphocytes were restimulated with peptide-pulsed irradiated autologous DCs

in

medium

containing

10

ng/mL

IL-7

and

10

ng/mL

IL-10,

and

then

supplemented

with 20 IU/mL IL-2 24 hours later. Lymphocytes were restimulated each week in the

same manner. At 7 days after the fourth round of restimulation, cells were harvested

and

tested

by

ELISPOT

assay,

cytotoxicity

assay,

and

tetramer

staining.

CD8T

lymphocytes

were

purified

by

CD4_

cell

–

negative

depletion

using

human

CD4

microbeads.

---------

Wang B, Chen H, Jiang X, Zhang M, Wan T, Li N, Zhou X, Wu Y, Yang F,

Yu

Y

et

al

:

Identification

of

an

HLA-A*0201-restricted

CD8+

T-cell

epitope

SSp-1

of

SARS-CoV

spike

protein.

Blood

2004,

104

(1):200-206.

第二军医大

曹

雪涛

5.

抗原肽诱导

CTL

制备：

密度梯度离心法分离经

EDAT

抗凝的静脉血中的外周血单个核细胞（

perip heral

blood

mononuclear