Adoptive transfer of bryostatin 1-activated T cells provides long-term protection from tumour metastases

Surgical Oncology 1992; 1: 299-307

299

Adoptive transfer of bryostatin l-activated T cells provides long-term protection from tumour metastases T. M. TUTTLE*, T. H. INGET, D. S. LIND* AND H. D. BEAR*? Departments

of *Surgery, tMicrobiology and Immunology, and The Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298, USA

Treatment frequent toxicity

of human

cancer with

unavailability

tumour-specific

of autologous

associated with high-dose

we demonstrate

that

Bryostatin

tumour

T lymphocytes

to stimulate

interleukin-2

(IL-2) treatment.

‘l (B) plus ionomycin

is limited

T-cell growth

by the

and by the

In the present study

(I) can substitute

for tumour

antigen and activate tumour-bearing hosts’ T-cells which provide long-term protection against tumour challenge after adoptive transfer. Lymphocytes obtained from the popliteal lymph nodes (DLN) draining an MCA-105 footpad sarcoma were stimulated with B/I, and then cultured for 7 days with 20 U ml-’ IL-2. This in vitro stimulation protocol consistently expanded cell numbers greater than 20-fold during 7 days. Mice given B/l-stimulated

draining lymph node (DLN) cells were protected from specific i.v.

tumour challenge for at least 15 weeks after adoptive transfer, even in the absence of IL-2 treatment. Tumour immunity conferred by B/l-activated DLN cells was systemic and independent of host T-cells. However, resistance to tumour challenge was lost when either CD4+ or CD8+ T-cells were depleted in who. These studies indicate that DLN cells activated with bryostatin 1 and ionomycin persist long-term in viva as functional memory cells after adoptive transfer. Surgical adoptive immunotherapy,

Keywords:

1992; 1: 299-307.

Oncology

bryostatin 1, effector cells, protein kinase C.

INTRODUCTION

stimulators

[3-51.

Therefore,

alternate

methods

of

activating T-cells are needed to broaden the applicaThe clinical application requires

the transfer

lymphocytes lymphoid

to tion

from

cancer

strategies

sensitized

lymphocytes

protocol

yields

T-cells

patients’

own

have been used T-cells for approach

with

Although which

bility

of activated

One effective

cells and IL-2 [I, 21.

metastases

immunotherapy

the growth of tumour-specific

immunotherapy.

culture

tumour

obtained

tissue. Several

to stimulate adoptive

of adoptive

of large numbers

is

irradiated

this stimulacan

eliminate

in vivo, this method may not have broad

clinical application

because

difficult

to

procure

because

human tumour

Correspondence:

from

tumour human

cells are often cancers,

and

cells may be poor immune

Dr Bear. Present address: Division of Surgi-

of

immunotherapy

adoptive

for

clinical

purposes. One

alternative

antibody

(mAb)

complex

to mimic

ever,

T-cell

limited,

has

been

to the

antigenic

growth

with

antigen

and the T-cell intracellular

the

ligand

this

results

membrane

how-

may

may actually

receptor

stores

(TcR)/CD3

binding;

approach

be

make

between in protein

and the release of calcium [8-IO].

of PKC and intracellular

T&f/CD3

monoclonal

[6, 71. The interaction

kinase C (PKC) activation activation

use

receptor

and too much antibody

T cells unresponsive

from

to

T-cell

complex

each arm of the T-cell activation

Pharmacological calcium bypasses and

stimulates

pathway.

Bryostatin

cal Oncology, Box 11, The Medical College of Virginia, Rich-

1, like some phorbol esters, is a potent PKC activator

mond, VA 23298. Phone: (804) 786-9323.

and

also

possesses

anti-neoplastic

properties

300

T. M. Tuttle et al.

[I I-131. either

We

have

demonstrated

previously

phorbol

dibutyrate

[I41

plus a calcium

ionophore,

in the absence

antigen,

can activate

are capable

tumours.

bypass

regression

Thus,

The

clinical

would

usefulness

be further

survive specific

long-lasting

vious investigators

reported

prepared

small fragments

[I 61.

cells

could

and provide memory.

Pre-

investigators

lymphocytes

challenge

(TIL) the

for up to 6 weeks

In the present murine

study, lymphocytes

(DLN) draining

MCA-105

bryostatin

sarcomas

After

obtained

progressively were

1 and the calcium

(B/I-DLN).

ionophore

in vitro culture

with

ionomycin

and growth

in low-

to sublethally

hosts. We found that T-cells activated

B/I could provide long-lasting tion

from

growing

activated

dose IL-2, these cells were transferred irradiated from

i.v.

tumour

deoxyribonuclease,

by mincing

hind footpad

solid tumours containing

(Sigma C57Bl/6 x

0.05 ml Hanks’

into

4 mg of

40 mg of collagenase,

with 5

(s.c.)

suspen-

by digestion with constant

Chemical

For establishing

mice were

inoculated

IO5 MCA-105

balanced

and 100 U

Co., St. Louis,

MO) for 3 h at room temperature. solid tumours,

tumour

Single-cell

stirring in 40 ml of RPMI-1640

sarcoma

salt solution

in a

cells in

(HBSS;

Bio-

fluids, Rockville, MD).

Lymphoid

cell suspensions

lpsilateral

popliteal

pad (DLN) were tumour

after adoptive transfer [I 71. lymph nodes

storage,

in viva and retain

Recently,

antigen and IL-2 protected

i.v. tumour

from

mice.

followed

of hyaluronidase T cells

that antigen-driven

that tumour-infiltrating

cultured with tumour

agents

for tumour

if these

have found

activity

host from

sions were

IL-2 treatment

T-cell clones can persist long-term anti-tumour

T cells which

T cells.

immunological

thawing

for passage by subcutaneous

of established

of B/l-activated

enhanced

in vivo without

After

in syngeneic

substitute

antigen and activate tumour-specific

respectively.

was transplanted inoculation

pharmacological

the TcR may

that 1 [15]

of tumour

tumour-specific

of mediating

metastatic which

or bryostatin

immunological

metastases

with

protec-

and

footpad

pared

injection. by

Single-cell

pressing

the

nodes draining

the foot-

10 days after

MCA-105

suspensions

nodes

were

through

pre-

IOO-mesh

stainless steel screens with the blunt end of a 3 ml plastic syringe plunger in HBSS under sterile conditions. The cell suspension suspended

in

Complete with

(Hyclone,

was washed

complete

medium

10%

medium

was

Logan,

amino

penicillin,

10 mM HEPES

acids,

bovine

and

5

X

serum

pyruvate,

0.1

2 mM L-glutamine,

100 ,ug ml-’

buffer,

and

culture.

of RPM1 1640

fetal

UT), 1 mM sodium

mM non-essential

twice,

before

composed

heat-inactivated

100 U ml-’

tumours, even in the absence of IL-2 therapy.

lymph

harvested

streptomycin,

10e5

M

2-mercapto-

ethanol (Sigma). MATERIALS

AND

METHODS Reagents

Mice

Bryostatin

Virus-free weeks

female

old,

Dawley

C57Bl/6

were

obtained

(Indianapolis,

BG.PL Thy-la/CY caged

IN).

(Thy

Jackson Laboratories were

mice

l.l),

(Thy

from Thy-l

1.2),

8-12

Harlan-Sprague congenic

were

obtained

of five or fewer

University, ionomycin,

Tempe, was

IO-*

by George

inverte-

previously

and

Pettit, (Arizona State

AZ) [18]. The calcium ionophore,

purchased

from

Calbiochem

1 and ionomycin

M stock solutions

man-LaRoche

from the marine

as reported

Diego, CA). Bryostatin recombinant

given food and water ad libitum.

neritina

was kindly provided

from

and were

1 was isolated

Bugula

mice,

(Bar Harbor, ME). The animals

in groups

brate

in DMSO

(San

were kept as

at -20°C.

Human

IL-2 (rlL-2) was kindly supplied

by Hoff-

(Nutley, NJ).

Tumours The MCA-105

and 203 sarcomas

threne-induced kindly

are methylcholan-

tumours

of C57Bl/6

by

Michael

provided

Cancer

Institute,

Chang

(University

Dr

Bethesda, of

MD)

Michigan,

Bryostatin

origin and were

Freshly

Lotze

were

and Ann

(National Dr

Alfred

Arbor,

Ml),

IAonomycin

harvested

incubated

DLN

cells at 1

x

with 5 nM bryostatin

mycin, and 20 U ml-’ fied air with

stimulation IO6 cells ml-’ 1, 1

,UM

iono-

IL-2 for 18 h at 37°C in humidi-

5% CO,. The cells were

then washed

Bryostatin l-activated T cells provide long-term memory three times with warm cultured

(37°C) HBSS. DLN ceils were

for 7 days in 300 ml tissue

(Baxter,

culture

Deer-field, IL) at 2.5 X IO5 cells ml-’

bags

with 20

U ml-’

IL-2. Cultures were split and refed with fresh

media

and IL-2 every 2 days. This in vitro stimula-

tion

protocol

greater

consistently

expanded

cell numbers

showed

elimination

DLN) were

administration was

found

of anti-Thy to

populations

deplete

adoptively

transferred

to hosts after 7

subset

of

Similar

1 .I or anti-Thy

1.2 mAb

splenic

T-cell

than 75% and 90%.

respec-

tively.

Phenotypic analysis

carried

Adoptive transfer studies Animals

received

500

from a 13’Cs source Canada,

rad whole

body

Canada).

Atomic

of 1 X IO’

cells in 1.0 ml HBSS. In some experiments, were

tumour

injection,

monary

nodules

insufflated

with 4 x IO5 tumour

points. the

mice

were

with

India

and

bleached

described

[19].

Metastases

too

arbitrary number

surface

The in

form

and

pul-

lung the

Fekete’s white

to count

of 250

of nodules

killed,

ink

with

days after i.v. were

trachea,

were

because

assigned

this is the

animals

recorded.

were

Mice

observed

of recurrent

were

was that

for

disease

fraction of mice without

an

largest enumer-

challenged

Hybridomas against

measured rejected

up to 3 months

CD4

American

CD3

Type

The mAb were tane-primed received

In all experiments,

(GK1.5),

profiles

containing

NaN,.

were

1%

Appropriate

antibodies

Non-viable

were

cells were

iodide staining. Fluor-

displayed

fluorescence

as logarithmically

intensity

versus

cell

Statistics The significance

Culture produced

were

murine obtained

Collection

CD8

mice treated

RESULTS Specificity of tumour immunity Draining

lymph node cells activated

analysis anti-CD4

with bryostatin

and cultured in 20 U ml-’ transferred

Mice

from MCA-105

tumour-bearing

the MD). mice

i.v. 2 days of spleen or anti-CD8

against

MCA-105,

challenge

(Experiment

experiment, T-cells MCA-203 against

but

the

obtained

adoptive

cells

obtained

MCA-203,

transfer

lymph

conferred

tumour specificity

of

nodes

but not against

ment 2). In contrast

cells

1 and 2,

hosts were protected

not

adoptive

sarcoma MCA-203,

B/I-DLN

1). In a crossover

from

mice.

with tumour

respectively).

given

IL-2 for 7

to irradiated

(2.43),

and again i.p. on the day of with

rank sum test.

i.v. 1 or 5 weeks later (Table 1, Experiments

from

(Rockville,

ascites

untreated

by the Wilcoxon-

(mAb)

as ascites fluid from pris-

Phenotypic

between

These mice were then challenged antibody

nude mice. For in vivo depletion,

before tumour challenge

of differences

mice was determined

1 and ionomycin

murine Thy 1.1 (TllD7e2),

0.25 ml of hybridoma

cells from

controls.

days were adoptively

monoclonal

(OKT3)

challenge.

(PBS)

and 0.1%

mAb red-free

for

as the

experiments

murine Thy 1.2 (30H12), human

escence

in phenol

with

tumour

and recorded

tumours.

producing

murine

for 45

Every

at least five mice were included in each group. ln vivo depletion

and

CA). Single-cell incubated

CA)

gated out based on propidium

numbers.

thickness

evidence

View,

isotype-matched

as negative

increasing

2-3

challenge

staining

microfluorometer

were

saline

serum

FITC-conjugated used

Diego,

buffered

and treated

footpad

San

mouse

on

cells in the hind footpad.

and

phosphate normal

nodules

that could be reliably

experiments,

days

Mountain

(1 X IO6 cells)

(Pharmingen,

as

with 5X IO5 tumour calipers

flow

was

min at 4°C with 1 ,ug of fluorescein-conjugated

solution

ated per mouse. In other

phenotypes

of the lung. Mice with meta-

numerous

value

were

counted.

15%

harvested

the blackened

Fourteen

cells

surface

FACScan

(Becton-Dickinson,

Within 4 h of irradia-

cell

immunofluorescence

a

suspensions

B/I-DLN

time

by

on

Energy of

given tail vein injections

challenged

of

irradiation

animals

i.v. at various

out

analysis

(Gammacell,

Ltd., Ottawa,

tion, mice were

tumour

appropriate

after treatment.

appropriate

by greater

Determination

and

of the

T cells for at least 2 weeks

than 20-fold during 7 days. These cells (B/I-

days in culture.

stases

301

specific MCA-105

to B/I-activated

transfer of fresh non-cultured

activated

draining

an

immunity (Experi-

DLN cells, the DLN cells did

T. M. Tuttle et al.

302

Table 1. B/I-DLN cells provide specific immunological protection from i.v. tumour challenge after adoptive transfer Cells given*

Pulmonary metastasest

Experiment 1 None B/I-DLN (MCA-105) Experiment 2 None B/I-DLN (MCA-203)

MCA-105$

MCA-203

250 (0)

250(O) 250(O)

O(O)5

not

protect

the

host

from

1

250(O) O(O)5

cells survive cells

Mice were

injected

after

ascertain

long-term

with

adoptive

whether

long-term

tumour

B/l-activated

B/I-DLN

cells

could

protection

for at least

15-23

to

confer

(Table

cells remained

1 and 2, respectively)

genie

were

2).

weeks

(Experi-

after the administration

performed

and the sites transferred

mice

received

were

Thy

to determine

of in viva trafficking

cells. Irradiated used

1.2 B/I-DLN

as

cells

typic

with

hosts

i.v. Seven

fluorescein-conjugated

analysis

majority

demonstrated

of splenic

that

lymphocytes

the of

Thy 1.1 con-

adoptive

mAb.

and

Pheno-

229(34) 250(O) 250(O)

O(O)5 132(92)§ 46(54)§

*Irradiated (500 rad) C57BV6 mice received 1 X 10’ B/IDLN cells i.v. and were then challenged with tumour cells i.v. at various times thereafter. Two weeks after tumour injection, mice were killed and lung metastases counted. tB/I-DLN: Lymphocytes were obtained from the popliteal lymph nodes draining an MCA-105 footpad tumour, stimulated with 5 nM bryostatin 1 and 1 ,~UM ionomycin, and then cultured for 7 days with 20 U ml-l ML-2 (B/I-DLN). *Each value represents the mean of at least five animals per group. Standard deviations are shown in parentheses. §Significantly different from untreated groups (P< 0.05, Wilcoxon rank-sum test).

transferred

Thy 1.2+ T cells could not be found

the

or

lungs

livers

of

these

animals

(data

Tumour

immunity

lungs

by FACS,

is systemic

conferred

by the local trapping

ferred

cells’in

However,

tumour

of adoptively

trans-

mice were

chal-

cells at a site distant from the

lung. One week or 5 weeks after adoptive transfer of B/I-DLN

cells,

mice

received

MCA-105

tumour

of host (Thy

exhibited

progressive

adoptively

cells in the that

but instead was simply

the lung. Therefore,

lenged with tumour

tumour-bearing

1).

still possible

was not systemic,

transfer

(Fig.

it was

immunity

1.2+

T-cells

in not

shown).

1.1) origin, 10% of the viable spleen cells were Thy donor

O(O)5 O(O)0 O(O)9 O(O)5 O(O)9 4(5)9 29(66)§

2

the

although

were

and

weeks

later, the spleens of these mice were harvested stained

250 (0) 243(16) 250(O) 208(24) 250(O) 232(23) 250(O)

Despite the inability to detect transferred

Experiments adoptively

2 3 5 7 10 15 Experiment 5 15 23

resistant

of DLN cells. persistence

B/I-DLN

cells i.v. at various of

lymphocytes

immunological given

as functional

transfer

these

to i.v. metastases ments

i.v. tumour

(data not shown).

memory

Animals

subsequent

MCA-105 pulmonary metastases* Cells transferred?

1

Experiment 250(O) 230(45)

B/I-DLN

times

Week of tumour challenge

Nonet

*B/I-DLN: Lymphocytes were obtained from the popliteal lymph nodes draining an MCA-105 (Experiment 1) or MCA203 (Experiment 2) footpad tumour, stimulated with 5 nM bryostatin 1 and 1 ,~UM ionomycin, and then cultured for 7 days with 20 U ml-’ IL-2. tlrradiated (500 rad) C57Bl/6 mice received 1 x IO7 B/I-DLN cells and were then challenged with tumour cells i.v. 1 or 5 weeks later in Experiments 1 and 2, respectively. Two weeks after tumour injection, mice were killed, and lung metastases counted. $Each value represents the mean of at least five animals per group. Standard deviations are shown in parentheses. SSignificantly different from untreated groups (P
challenge

Table 2. Adoptive transfer of B/l-stimulated DLN cells protects the host from i.v. metastases for at least 15 weeks

cells

tumour

of B/l-stimulated mice

(Fig.

footpad 2).

injections

Untreated

growth,

DLN cells from

protected

while

of

mice the

MCA-105

the adoptive

hosts

Bryostatin

l-activated

T cells provide

long-term

memory

303

100 -

Figure

1. FACS analysis

splenocytes weeks

of fresh

from Thy 1 .I host mice 7

after i.v. injection

cells obtained Although were

from

of B/I-DLN

Thy 1.2 donors.

the majority

of lymphocytes

of host (Thy 1 .l ‘) origin,

population

of donor

a small

(Thy 1.2+) T cells

was clearly identified.

Figure ?. Footpad and 5 weeks

means

Values

mice were shown

and standard

footpad

challenge

after i.v. injection

DLN cells. Normal controls.

tumour

thickness.

deviations

tumour-free

mice at 3 months

challenge of animals

0-b

from

the

the number

as a fraction injected

of

after of total

with Days after

footpad

tumours.

B/I-DLN Control

of

tumour.

were tumour-free

o--o

in

indicate

number

Week 5

used as

parentheses tumour

I

of B/I-

represent

Values

Week

1

All

mice

given

B/I-DLN

cells

for at least 3 months after footpad

tumour challenge.

immunological DLN

cells

antibody,

footpad

injection

protection

was

against metastases

abrogated.

anti-human

An

irrelevant

by B/Icontrol

CD3, had no effect on tumour

resistance. Protection and CD8’ Depletion

from metastases

requires

both CD4+

T-cells

Immunity

of T-cell subsets was performed

tain whether

specific

immunity

required

CD8’

T-cells (Table 3). When either anti-CD4

CD8

monoclonal

C

antibody

was

given

to ascerCD4+

or

or antiin

vivo,

Next,

we

necessary cells

is conferred determined

whether

for resistance

obtained

MCA-105

by donor T-cells

from

Thy

host

to tumour

T-cells

challenge.

1.1 congenic

footpad tumours were activated

mice

were DLN with

with bryo-

304

T. M. Tuttle et al. Table

Cells given*

mAb givent

MCA-105

pulmonary

metastases$ Exp I§

Exp 2

229(34)

None

B/I-DLN

None

O(O)lf

250 (0) -

B/I-DLN

0KT3

B/I-DLN

anti-CD4

O(O)7 228(31)

241(16)

B/I-DLN

anti-CD8

221(28)

243(14)

*B/I-DLN:

Lymphocytes

ionomycin, tMAb

were obtained

footpad tumour,

from i.v.

requires both CD4+ and

CD8+ T cells

None

an MCA-105

3. Protection

metastases

17(21)ll

from the popliteal lymph nodes draining

stimulated

with 5 nM bryostatin

and then cultured for 7 days with 20 U ml-’

1 and 1

,LdM

IL-2 (B/I-DLN).

was given as 0.25 ml ascites i.v. 2 days before and again i.p. on the day

of i.v. tumour injection. SIrradiated (500 rad) C57BV6 mice received then challenged injection,

mice were sacrificed

§Each value represents deviations

1 x lo7 B/I-DLN cells i.v. and were

with tumour cells i.v. 2 weeks later. Two weeks after tumour and lung metastases

counted.

the mean of at least five animals per group. Standard

are shown in parentheses.

llsignificantly

different

from untreated

groups (PC 0.05, Wilcoxon

rank-sum

test).

Table 4. Protection upon the presence

from tumour challenge of adoptively

is dependent

transferred

donor T cells

Cells

In vivo

MCA-105

transferred*

mAbt

metastasest

None

-

B/I-DLN

OKT3

B/I-DLN

anti-Thyl.2

B/I-DLN

anti-Thy1 .l

however,

in viva depletion

abrogated

immunity

of donor (Thy 1 .I ‘) cells

to tumour challenge.

pulmonary

DISCUSSION

*DLN cells obtained were stimulated

131(49)§

One limitation

O(O)JJ 9(9)lf 142(67)

with T lymphocytes stimulate

from Thy 1.1 tumour-bearing

with 5 nM bryostatin

1 and 1

mice

of large numbers

that mice with established

metastases

the adoptive

transfer

again i.p. on the day of tumour challenge. weeks after adoptive

transfer

of B/I-DLN cells, mice

were given i.v. injection of MCA-105 §Each value represents per group. Standard llsignificantly

different

rank-sum

are shown in parentheses.

from untreated

cate

that

survive

DLN

were

groups (P< 0.05,

test).

animals immunity

cells

expanded

specific

given

0KT3,

after

tumour-drain-

adoptive

B/I-DLN

weeks

later,

anti-Thy

these

cultured mice

1.1 or anti-Thy

for 7 days in IL-2, received

irrelevant

1.2 mAb and tumour

cells (Table 4). OKT3 and anti-Thy no effect on the number

cells

vitro

transfer

with

and

B/I

retain

activity.

Moreover,

developed

systemic

without IL-2 treatment;

no cytokines were

given either with the lymphocytes

or at the time of

tumour challenge.

into the tail veins of Thy 1.2 mice.

injected

in

anti-tumour

Previous investigators

Two

cured of tumour

of B/l-activated

in viva after

long-lasting

tumour cells.

the mean of at least five animals

deviations

statin 1 and ionomycin, and then

of tumourOur previous

ing lymph node cells [15]. The present studies indi-

was given as 0.25 ml ascites i.v. 2 days before and

Wilcoxon

pulmonary

cancers

is that tumour cells necessary to

the growth

work has demonstrated

,a~

(Thy 1 .l).

*Two

of human

specific T cells are often not available.

ionomycin and cultured for 7 days. Irradiated (500R) Thy 1.2 mice received an i.v. injection of 1 x 10’ B/I-DLN cells tMAb

to the treatment

1.2 antibody

of pulmonary

had

metastases;

have also demonstrated T cells

can

documented

survive

working

with other models

that adoptively

long-term

that virus-specific

transferred

in vivo. CD8+

Jamieson

T-cells

could

persist for the life span of the host as functional memory the

T cells [20].

antigen-driven

Studies

by Cheever

long-term-cultured

indicate

T cell

lines

Bryostatin l-activated derived

from

immune

mice can proliferate

rapidly

and survive

long-term

in vivo, and retain

specific

anti-tumour

activity

Alexander

has recently

with tumour

antigen

in viva after diated

after

adoptive

reported

transfer

that TIL cultured

transfer

hosts or to animals

to sublethally

with

are

obtained

unique,

from

hosts and were logical

because

the lymph

agents

this non-specific cells provided demonstrated

tumour

of tumour-specific vitro activation

cells [I,

antigen

cells does not provide i.v. tumour challenge The animals

Although

However,

lymphoid

preliminary

cells activated vide

we have found harvested

from

DLN

subsequent

mycin

has

include

the

mately

80%

CD8’

culture in low-dose

by the transfer

CD4+

and

CD8+

found that when

pro-

were

the

to

combined cate

the potential

approxi-

after

7 days

where

to

they

the protect

or

the

Mice congenic to

determine

dependent

upon transferred

T cells. Administration antibody

tumour

donor cells and/or

of anti-Thy

to Thy 1.2+ hosts abolished

logical protection

induced

long-term

by the donor (Thy 1 .I ‘)

of host (Thy 1.2+) T cells.

is in agreement

with

observations

on the long-term

memory

transferred

Our findings

the

vant

have

significant 1

lymphocytes retrovirally

and

of

of both CD4+

impliand

is

by these toxic

IL-2

in viva or to retain anti-

are a proposed

in the adju-

cancers. vehicle

Moreover,

for delivering

genes to patients with cancer

have

here

antigen

activated

investigators

both

be for

do not require

human

transduced

may T cells

tumour

1 and ionomycin

mice,

of

relevance

ionomycin

when

agents

ideal means of stimulating for the administration

function

clinical

tumour-specific

Since lymphocytes

treatment

published

TIL [ 171.

bryostatin

not available.

transferred

memory func-

finding

adoptively

host

the immuno-

by adoptively

Thy 1 .I + T cells. Therefore,

was

1.1 monoclonal

diseases

120 days after adop-

from

immunity

T cells with bryostatin

T-cells

host

for the Thy 1 antigen were utilized whether

both

cultured

footpad,

tumour development.

cells required

of other investigators

participation

lung

or immunodeficiency

[16]. The results reported

with findings

sites

con-

tumour-bearing

CD4+ and CD8+ cells survived tive immunotherapy

respectively,

Howthese

can traffic from the spleen or pos-

other

immunity

antigen-stimulated

transferred

from

challenge,

tumour function, they may be applicable

were

adoptive

after transfer.

tumour

lymph node cells

CD4+

Other

that cultured after

pharmacological

cells

of B/I-DLN

vivo

to non-irradiated

1 and iono-

may over-

but not the lungs or livers,

therapy to persist long-term

20%

T-cells.

cells apparently sibly

immunotherapy

IL-2 [15]. Tumour

ferred

in

hosts 7 weeks

adoptive

indicate that DLN

1 and ionomycin

with bryostatin

and

in the spleens,

to activate

function

that

identified

of the adoptive

because

cells [25].

of

trans-

[29, 301. In our studies, donor T cells were

employed

analysis of draining revealed

abnormally

for donor

experiments

after in vitro stimulation

traffic

cells or the creation

hosts also (data not shown). Phenotypic

cells

This

trans-

challenge

Previous studies have demonstrated T

mechan-

‘space’

memory

of tumour

the precise

irradiation

for in vivo prolifera-

come the need for helper cell function.

cells and is independent

sublethal

The trans-

lymphokines,

cells. Administration

tion appears to be conferred

explanations

with bryostatin

long-term

functional

the survival of adoptively

Possible

fer or at the time

all given

of host suppressor

of increased

monoclonal

[26-281.

IL-2 either at the time of adoptive

study were

understood,

appears to enhance T-cells.

in

(data not shown).

irradiation.

elimination

protection

exogenous

freshly

require

to become

of freshly

in the present

ism is not clearly ferred

nodes

7, 151. Moreover, transfer

source

However,

lymph

have

a progres-

an enriched

with either B/I, anti-CD3

that the adoptive

sublethal

that was spe-

and others

T-cells [22-241.

or tumour

effector

provide

tumour-draining

antibody,

We

tion and survival of effector

ever, after i-v. or footpad

B/l-stimulated

immunity

tumour.

Despite

immunity

helper cells may provide

such as IL-2, that are essential

transfer

by pharmaco-

antigen.

that lymph nodes draining

sively growing harvested

in culture

of tumour

long-lasting

were

of tumour-bearing

method of activation,

cific for the original

meta-

CD8+ T-cells in tumour fer of CD4+

in the present

lymphocytes

nodes

expanded instead

irra-

established

stases [17, 211. The results reported studies

[I 61.

and IL-2 can persist long-term

adoptive

305

T cells provide long-term memory

[31-331.

the growth

Activating may be an

of these cells

of gene therapy to such adop-

tive hosts. This work was supported by grant CA48075 from DHHS, NCI. THI is a recipient of a Medical Scholars Award from the AD. Williams

Foundation

Norfolk Foundation.

and

a scholarship

award

from

the

T. M. Tuttle et al. 13. Pettit

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