graft-versus-leukemia reactivity involves cluster ... · parke, davis & co., berlin, germany),...

Vol. 4, 3095-3106, December 1998 Clinical Cancer Research 3095

Graft-versus-Leukemia Reactivity Involves Cluster Formation

between Superantigen-reactive Donor T Lymphocytes and

Host Macrophages’

Susanne M#{252}erk#{246}ster,Olga Wachowski,

Heide Zerban, Volker Schirrmacher,

Victor Umansky, and Marian Rocha2

Deutsches Krebsforschungszentrum, Abteilung ZellulareImmunologie, FSP Tummorimmunologie IS. M., 0. W.. V. S., V. U..M. R.] and Deutsches Krebsforschungszentrum, Abteilung

Cytopathologie, FSP Krebsrisikofaktoren und Krebsprhvention

[H. Z.], 69120 Heidelberg, Germany

ABSTRACT

T-cell-mediated antitumor effects play an importantrole clinically in allogeneic graft-versus-leukemia (GvL) re-activity, whereas T-cell-mediated antihost effects are associ-

ated with a risk of developing graft-versus-host (GvH) dis-ease. GvL and GvH were compared in an animal tumormodel system after the systemic transfer of allogeneic anti-tumor immune T lymphocytes from B1O.D2 [H-2�’; minorlymphocyte-stimulating antigen (Mls) bj mice into ESb-MP

tumor-bearing or normal DBA/2 (H-2�’; Ml?) mice. Here we

demonstrate that this T-cell-mediated therapy involves theformation of clusters of donor CD4 and CD8 T cells withhost macrophages, in particular, with a subpopulation ex-

pressing the lymphocyte adhesion molecule sialoadhesin.

DBA/2 mice and the derived tumor ESb-MP express viral

superantigen 7 (Mlsm), an endogenous viral superantigenthat is absent from B1O.D2 mice. To test the contribution ofviral superantigen 7-reactive Vf36 donor T cells in the GvL-mediated eradication of liver metastases, we performed im-munohistological and transmission electron microscopystudies. V�6+ CD4 and CD8 T cells from B1O.D2 donors

formed tight clusters with host sialoadhesin-positive macro-phages, and transmission electron microscopy pictures re-vealed direct membrane-membrane interactions between Tcells and macrophages. Clusters were more abundant and

consisted of more cells in tumor-bearing hosts (GvL model)than in non-tumor-bearing hosts (GvH model). In addition,V�6 T cells within the clusters showed a strong proliferationactivity, indicating stimulation. Moreover, in an in vitro

Received 7/3/98: accepted 8/26/98.

The costs of publication of this article were defrayed in part by the

payment of page charges. This article must therefore be hereby marked

advertisement in accordance with 18 U.S.C. Section 1734 solely to

indicate this fact.I Supported in part by the Dr. Mildred Scheel Stiftung (V. U. and M. R.).2 To whom requests for reprints should be addressed, at DeutschesKrebsforschungszentrum, Abteilung Zellul#{228}re Immunologie, FSPTummorimmunologie, Im Neuenheimer Feld 280, 69120 Heidelberg.

Germany. Phone: 49-6221-423708; Fax: 49-6221-423702; E-mail:[email protected].

tumor cytostasis assay, primed as well as nonprimed pun-fled Vf�6 T cells from donor mice were able to inhibit the

proliferation of superantigen-expressing ESb-MP lym-

phoma cells. This suggests that the transferred superanti-

gen-reactive V�6 T cells contribute to the eradication of

metastases. The observed cell clusters might be sites forantigen presentation and the activation of tumor-reactive Tcells.

INTRODUCTION

The transfer of activated tumor-reactive lymphocytes into

tumor-bearing animals has been reported as an effective ADI3

against clinically established metastases (1-3). T-cell-mediated

antitumor effects also play an important role in the so-called

GvL effect that is usually associated with a risk for the devel-

opment of GvH disease with significant morbidity and mortality

(4). We have established an animal model for the investigation

of the GvL and GvH reactivity of immune lymphocytes. The

murine leukemia system consists of various well-defined me-

tastasizing sublines (ESb, ESb-MP, and ESbLlacZ) that are

derived from the chemically induced DBA/2 lymphoma

L5 I 78Y. The AD! effects are achieved by the transfer of in

situ-activated tumor-reactive lymphocytes from the tumor-resis-

tant strain B l0.D2 into susceptible DBAJ2 syngeneic mice.

DBA/2 mice differ from Bl0.D2 mice not only in minor histo-

compatibility, but also in Mls antigens (5). These Mls determi-

nants represent vSAGs encoded by MMTV proviruses that are

integrated in the murine genome (6, 7). The possibility thus

exists that the extraordinary resistance of B l0.D2 (Mls�’) mice to

DBA/2 (Mls”) tumors may be due in part to a superantigen

difference. This possibility is supported by our recent findings

of a link between the absence of the endogenous MMTV super-

antigen vSAG7 and tumor resistance (5). vSAG7 behaves as a

self-tolerogen in DBA/2 mice by deleting superantigen-reactive

T cells with certain V�3 chains (e.g., V�36) from their repertoire

(8). We have recently found that ESb tumor cells express not

only individually distinct class I MHC (Kd)-restricted CTL

epitopes (5, 9) but also express proviral Mtv-7 at the RNA

level.4 One hypothesis to explain the mechanism of ADI is that

upon ESb tumor cell transfer into BbO.D2 mice, V�36+ T cells

may become activated by the tumor-associated vSAG and, after

3 The abbreviations used are: ADI. adoptive cellular immunotherapy:GvL, graft-versus-leukemia, GvH, graft-versus-host� MIs. minor lym-phocyte-stimulating antigen: vSAG, viral superantigen: MMTV. mouse

mammary tumor virus: Sn. sialoadhesin: TAA. tumor-associated anti-gem P0. horseradish peroxidase: AP, alkaline phosphatase: DC. den-dritic cells: Th, T helper: APC. antigen-presenting cell: NO, nitric oxide:mAb, monocbonal antibody: IL, interbeukin.

4 Unpublished observations.

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3096 Superantigen Reactive T Cell-Macrophage Cluster Formation during ADI

injection into tumor-bearing DBA/2 mice, may infiltrate ESb

liver metastasis. Other results from this model show that a

subset of host macrophages bearing the lymphocyte adhesion

molecule Sn is able to process exogenous TAA from metastases

and present it not only via MHC class II to CD4 cells (10) but

also via class I to CD8 immune donor T lymphocytes.5 These

macrophages could also present tumor-derived vSAG via their

MHC II molecules, because in t’ivo studies have provided strong

arguments for the paracrine transfer of vSAGs from cells that do

not express MHC class II to MHC class Il-positive cells profi-

cient in vSAG presentation (1 1).

Therefore, the aim of this work was to study the possible

physical interactions in situ between Sn+ host macrophages and

V�36 superantigen-reactive donor T cells in the liver of tumor-

bearing mice during ADI by immunohistology. We demonstrate

that upon transfer into tumor-bearing DBA/2 recipients, V�6+

CD4 and CD8 T cells from B b0.D2 donors form clusters a few

days later with host Sn+ macrophages in metastasized livers.

Tight membrane interactions between T cells and macrophages

in these clusters will be shown by electron microscopy. More-

over, we will show that purified nonprimed V�6 donor T cells

are cytostatic in vitro for ESb tumor cells, suggesting that there

are also in t’it’o antitumor effects after specific interactions of

V�36 T cells with a tumor-exposed superantigen.

MATERIALS AND METHODS

Animals

DBA/2 mice were obtained from IFFA Credo (Lyon,

France), and Bl0.D2 mice were obtained from Olac (Bicester,

United Kingdom). The animals were used at the age of 6-12

weeks.

Tumor Cell Lines

ESb cells represent a spontaneous highly metastatic variant

of the chemically induced T lymphoma L5 178Y (Eb) of DBA/2

mice. ESb-MP is an adhesion variant of ESb that grows in vitro

attached to plastic, whereas ESb grows in suspension. in vivo,

ESb-MP cells grow progressively but show a less aggressive

phenotype, metastasizing more slowly than ESb cells and in-

volving multiple organs (12).

Priming of B10.D2 Donor Mice and ADI of Recipient

DBA/2 Mice (GvL and GvH)

DBA/2 mice were anesthetized with Rompun (0.1%;

Parke, Davis & Co., Berlin, Germany), Ketanest (0.25%: Bayer,

Leverkusen, Germany), and PBS at a volume of 1 : 1 :3 to inject

2 X b0� ESb-MP tumor cells into the dermis of the shaved flank.

To generate albogeneic immune effector cells, ESb-MP cells

were injected iv. at a dose of l0� cells into the tail vein of

B 10.D2 mice. Seven days later, the spleen cells were isolated

and transferred at a dose of 2 X b0� cells!200 p.1 PBS into

S � MOerkdster. M. Rocha, P. R. Crocker, V. Schirrmacher. and V.Umansky. Sialoadhesin-positive host macrophages play an essential rolein graft versus leukemia (GVL) reactivity in mice: processing andpresentation of exogenous (tumor) antigen via MMC class I, submittedfor publication.

sublethally irradiated DBA/2 mice (5 Gy; 60Co source Gamma-

tron F 80 5; Siemens, Braunschweig, Germany) that carried

tumors (> 1 cm in diameter) 3 weeks after intradermal tumor

cell inoculation. Sublethally irradiated tumor-bearing DBA/2

mice in the control group remained untreated. To analyze GvH,

non-tumor-bearing DBA/2 mice were irradiated and inoculated

with the immune spleen cells.

Antibodies and Other Reagents

The following mAbs were used as culture supernatants: (a)

rat-antimouse CD8 (clone Lyt-2; Ref. 13); (b) rat-antimouse

V�6 (clone 44-22- 1 ; Ref. 14); (c) hamster-antimouse B7. 1

[clone 16-1OA)l; Ref. 15]; and (6) rat-antimouse Sn (clone

SER4; Ref. 16). The first antibodies were visualized by using

polycbonal donkey antirat and antihamster IgG (H + L) anti-

bodies linked to P0 and to AP (Dianova, Hamburg, Germany).

A biotinylated rat antimouse antibody was used (Life Technol-

ogies, Inc., Eggenstein, Germany) for the immunohistological

detection of CD4 T lymphocytes, and ExtrAvidin was linked to

AP (Sigma, Deisenhofen, Germany).

immunohistochemistry

Tissue Preparation. Livers were removed and snap-

frozen in liquid nitrogen. Six-p.m-thick consecutive cryostat

sections were mounted on uncovered glass slides, air-dried

overnight at room temperature, and fixed in acetone for 10 mm

at room temperature or air-dried for 1 h.

Single Staining. After fixation and drying, the slides

were washed in PBS three times for 5 mm. To avoid nonspecific

binding, the sections were treated for 20 mm with 1% normal

mouse or rat serum, followed by an incubation with the first

antibody for 45 mm. After washing three times with PBS, the

sections were incubated for another 45 mm with the second

antibody and washed before performing the substrate reaction

for P0 or AP. When using a biotinylated antibody, the slides

were incubated with ExtrAvidin for 30 mm. After staining, the

sections were washed with water, counterstained with hemalaun

(Merck, Darmstadt, Germany), and mounted with glyceryb gel-

atm (Merck). The same protocol was performed for negative

controls in which either the first or the second antibody was

omitted. All steps were performed in a humid chamber and at

room temperature.

Double and Triple Staining. The staining procedure

represents a combination of consecutive stainings for each an-

tigen. Every single staining was completely finished with the

substrate reaction before starting the second and third staining

procedure, respectively. No counterstaining with hemalaun was

performed.

Development of Enzyme Reactions. P0 activity was

revealed by incubating the sections in a solution containing 6

mg of 3-amino-9-ethylcarbazole (Merck) dissolved in 1.5 p.1 of

N,N-dimethylformamide (Merck), 15 p.1 of 30% hydrogen per-

oxidase, and 28.5 ml of 0.1 M acetate buffer (pH 5.0). The

substrate for the development of AP consisted of 6.3 p.1 of 5%

Neufucsin (Sigma) or 2 mg ofFast Blue (Sigma) in 16 p.1 of 4%

sodium nitrite (Fluka, Buchs, Switzerland), 2 mg of naphthob

AS-B! phosphate (Sigma) in 20 p.1 of N,N-dimethylformamide,

and 3 ml of 0.05 mob/biter Tris-HC1 buffer (pH 8.7) containing



Clinical Cancer Research 3097

1 mM Levamisole (Sigma). The freshly prepared solutions were

filtered and added to the sections. Development lasted about 3-5

mm, with regular checking of the reaction intensity by micros-

copy. Immunohistochemical results were evaluated by counting

the number of positively stained cells per liver lobule from three

to six lobules/mouse. The means and SDs of the data obtained

from two to three mice/time point were calculated and presented

in the figures.

Electron Microscopy

On day 12 after ADI, DBA/2 mice were sacrificed, and

liver samples of each ( 1 mm3) were cut and fixed immediately

in 2.5% glutaraldehyde buffered with 0.05 M cacodylate and

supplemented with ions (50 mrvi KC1 and 2.5 mrvi MgCI,) for 30

mm at 4#{176}C.After washing several times with 0. 17 M cacodylate

buffer, the samples were postfixed with 2% osmium tetroxide in

0.1 1 M cacodybate buffer for 2 h at 4#{176}C.After washing several

times with distilled water, samples were dehydrated with graded

series of ethanol and propylene oxide and embedded in a mix-

ture of Araldite and Epon resin. Ultrathin sections were cut and

double-stained with a 1.5% ethanobic solution of uranylacetate

for 20 mm and with lead citrate as described by Reynolds (17)

for 3 mm.

Flow Cytometric Analysis

Spleen cells (1 X 106) were washed in PBS supplemented

with 5% FCS and treated with mAbs against mouse Fc receptors

to avoid nonspecific binding, and then the staining for surface

molecules was performed, which included the following anti-

bodies: (a) F!TC-labeled anti-V�36; (b) phycoerithrin-labeled

anti-CD4; and (c) Red 613-labeled anti-CD8 (PharMingen,

Hamburg, Germany). Intracellular staining for cytokines was

performed as described previously (18). Briefly, after fixation in

4% formaldehyde (Merck), the cells were washed in PBSIFCS

buffer containing 0.5% saponin (Sigma) and stained with one of

the following FITC-conjugated mAbs: (a) anti-IL-2; (b) anti-

IL-4; or (c) anti-IFN-’y (PharMingen). After washing once in

0.5% saponin-containing buffer and once in PBSIFCS, the cells

were analyzed by flow cytometry using a FACScan with

CELLQuest software (Becton Dickinson, Heidelberg, Germa-

ny). Typically, 30,000 events were collected, and the data were

expressed as dot plots.

Isolation of V�6+ T Cells

BlO.D2 mice were primed with 5 X l0� ESb-MP cells

injected intra-ear. After 7 days, single-cell suspensions from the

spleens of primed and nonprimed mice were prepared by me-

chanical dissociation. After the lysis of erythrocytes by a short

NH4C1 hypotonic solution treatment, residual cells were washed

twice and resuspended in PBS with 2% FCS. Cells were incu-

bated with an anti-V�36 mAb (100 p.1 supernatantll06 expected

V�36+ cells) for 15 mm on ice. After two washing steps, the

cells were labeled with 60 p.l/l07cells of magnetic bead-conju-

gated antirat IgG isotype antibodies (MACS; Miltenyi Biotec

GmbH, Bergisch Gladbach, Germany) in 250 p.1 of PBS and 2%

FCS for 15 mm on ice. After washing, cells were collected in the

column buffer (PBS containing 5 m�vi EDTA and 0.5% FCS).

The depletion of labeled cells was achieved by passing the cells

through a steel wool-containing MACS column that was at-

tached to the MACS magnet ( 19). V�36- cells were washed out

by an excess of column buffer, whereas the labeled V�36+ cells

remained in the column. To obtain the positive selected cells,

the column was removed from the magnet, and the cells were

washed out with column buffer. To prove the purity of the

positively selected cells, they were incubated with a FITC-

conjugated anti-V�36 mAb (Life Technologies, Inc.) and meas-

ured by flow cytometric analysis. Cells were also stained with

1 .5 p.mol/liter propidium iodide to exclude dead cells. Flow

cytometric analysis was performed using a FACScan (Becton

Dickinson). Excitation laser frequency was 488 nm, and fluo-

rescence emission was detected at 575 nm. Typically, 10,000

events were collected and analyzed.

Cytostasis Assay

ESb-MP tumor target cells were resuspended in medium

(RPMI 1640: Life Technologies, Inc.) with antibiotics, 10% heat-

inactivated FCS, 2 msi glutamin, 10 msi HEPES, and 50 p.M

2-mercaptoethanob, and 5 X l0� cells!20 p.1 medium/well were

seeded in sterile 96-well round-bottomed plates (Renner,

Dannstadt, Germany). To promote central adherence, the cells were

incubated at 37#{176}Cfor 2 h. Effector cells (V�36+ and V�36- spleen

cells from both primed and nonprimed mice) were added in a

volume of 180 p.1 with a final E:T ratio of 10: 1. After 48 h of

incubation at 37#{176}C,1 p.Ci of [3Hjthymidine (Amersham, Braun-

schweig, Germany) was added per well. Incorporated radioactivity

was measured after 16-20 h in a liquid scintillation beta counter

(LKB Wallac, Freiburg, Germany). Values from the experimental

and control wells were compared to calculate the percentage of

proliferation and the growth inhibition of tumor cells.

RESULTS

Induction of Cytokine Production in the Spleen Cells ofB1O.D2 Mice upon Immunization with Tumor Cells. First

we studied the production of cytokines in the GvL effector cells

of our model system by flow cytometry. Seven days after

ESb-MP tumor cell injection, the spleens of BlO.D2 mice were

stained intracellularly with antibodies against IL-2, IL-4, and

IFN-�y. Fluorescence-activated cell-sorting analysis of total lym-

phocytes revealed that the number of IL-2-, IL-4-, and IFN-y-

producing cells was 2-3-fold higher in primed mice than in

nonprimed (control) mice (Table 1). To evaluate the contribu-

tion of the specific superantigen-reactive V�6 T cells to cyto-

kine production, double staining was performed with a mAb

against the V�36 surface molecule, and intracellular staining was

performed with anticytokine mAbs. As shown in Table 1, there

was a large increase in V�36 IL-2-producing (10-fold) and IFN-

-1’-producing (8-fold) cells upon immunization, thus pointing

toward a Thl-type cytokine response profile. V�36 T cells rep-

resented about 10% of the donor T-cell population.

Eradication of Lymphoma Liver Metastasis after ADI

as Visualized by the Disappearance of Ki67+-stained Met-

asiatic Foci. B l0.D2 anti-ESb-MP immune spleen cells, as

described above, were transferred into ESb-MP tumor-bearing

DBA/2 mice irradiated with 5 Gy. To evaluate the efficiency of

such an ADI, livers from treated animals were removed 2 and 20

days after immune T-celb transfer (early and bate stage) and



Group IL-2

Total lymphocytes

IL-4 IFN-y IL-2

Nonprimed 1.4 ± 0.2 0.1 ± 0.1 1.0 ± 0.1 3.3 � 2.0 3.1 ± 1.7 2.9 ± 1.9

Primed 4.0 ± 0.6 0.2 ± 0.1 1.9 ± 0.3 33.6 ± 3.3 6.7 ± 2.2 23.3 ± 2.4

V�36+ cells

IL-4 IFN--y

!J�r�i1. ‘ .-

m

pv

Fig. 1 Immunohistochemical picture of fro-zen tissue sections that were stained for pro-liferation marker Ki67 in the livers of tumor-

bearing mice at day 2 (A) and day 20 (B)after ADI treatment. Magnification, X 100.Pr, portal vein: m, metastases.

1�

a

.0

�v�#{149}

a


‘�.

Table I Number of cytokine-producing cells

Number of cytokine-producing cells (%)

..� � ., :..�� :. . . � � . .. . . . �, . . .. . S � : � �

� �‘ 4��M#{149}4� .. I � :�B .� . 4, .� S � ‘ .-..,.

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snap-frozen, and cryostat sections were stained with the prolif-

eration marker Ki67, which recognizes a nuclear antigen present

in proliferating cells (20). As shown in Fig. 1A, 2 days after the

transfer, livers from tumor-bearing mice contained groups of

proliferating tumor cells, which were spreading from the portal

vein into the parenchyma. At 20 days after therapy (Fig. 1B),

only some proliferating cells remained in the liver. These were

not in clusters, as were the metastatic tumor foci, and could

represent proliferating donor T cells. In addition to other pa-

rameters that have been reported previously (4), the disappear-

ance of tumor foci demonstrates the efficiency of this therapy.

CD4 and CD8 Donor T Cells Form Clusters with HostSn+ Macrophages. To distinguish between GvL and GvH

reactivity, we performed comparative studies in either tumor-



‘�,

day5 cLay2O

0.� 4r:d(

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Fig. 2 Immunohistochemical picture of frozen tissue sections that were triple-stained for CD8 (brown), CD4 (blue), and Sn (pink) in the livers of

tumor-bearing mice (GvL) at day 5 (A and B) and day 20 (D and E) after ADI therapy and in the livers of non-tumor-bearing mice (GvH) at day 5(C) and day 20 (F) after ADI. Magnification, X 100 (A and D) and X200 (B, C, E, and F). pv, portal vein.

bearing mice (the GvL situation) or non-tumor-bearing mice

(the GvH situation) after immune cell transfer. At different

times after i.v. immune cell injection, the livers of the recipients

were removed and snap-frozen to be analyzed later by immu-

nohistochemistry. The number of detected T cells was calcu-

bated as the mean cell count ± SD obtained per section of a liver

lobule. Triple staining with mAbs against CD4 and CD8 mob-

ecules for T cells and with mAb against Sn for Sn+ macro-

phages revealed the location of these cells in the liver sections

of mice undergoing AD! therapy (10). We observed that these

three types of cells formed tight clusters in which different

numbers of each cell type were present. We define cell aggbom-

erates containing at beast one cell of each of the cell types

described above as a cluster. Fig. 2 shows a representative



0 5 10 15 20 25 30

days after AD!


a)

d0

Fig. 3 Kinetics of CD8/CD4/Sn cluster formation in the livers of

tumor-bearing ([I]: GvL) and non-tumor-bearing ( #{149}; GvH) DBAJ2 miceafter ADI treatment. Frozen tissue sections were stained for Sn+ macro-

phages. and CD8 and CD4 T lymphocytes were stained with the cone-

sponding mAbs. Three to six lobules/liver were analyzed under themicroscope. Data show the means and SDs from two experiments with

two-three animals/time point. Data points without SD represent the

mean values for which the SD was smaller than 1.

image of cluster formation in the early (day 5) and late (day 20)

stages after immune cell transfer in GvL (A-C) and GvH (D-F).

The clusters started to appear in the liver 5 days after donor cell

transfer (2 1 ). They were located in the periportal areas close to

the blood vessels and in the parenchyma. Fig. 3 shows the

kinetics of cluster formation comparing the GvL and GvH

situations. In the early stages (day 5) in the GvL situation,

clusters were less abundant than in the later stages (day 20),

when around 10 clusters/liver lobule could be found. In GvH,

clusters contained smaller numbers of cells in both the early and

bate stages, and they disappeared from the liver around day 20.

The numbers of cells within the clusters also varied (Fig. 2) and

were highest in late-stage GvL. To elucidate cell surface mem-

brane-membrane interactions within the clusters, we performed

transmission electron microscopy studies 8 days after the im-

mune cell transfer. Fig. 4 shows a typical section through a

Kupffer cell (K) with its characteristic electro-dense cytoplasm,

variety of lysosomal structures, and inclusions in close mem-

brane interaction with two lymphocytes (L). Arrows point to

areas of membrane contact between Kupifer and lymphocyte

cells. Because Sn- macrophages hardly formed clusters with T

lymphocytes,5 we conclude that this macrophage-bymphocyte

interaction is a special property of Sn-expressing macrophages.

Cluster-associated Sn+ Macrophages Are Activated.

To investigate whether cluster formation might be a reflection of

an active antigen presentation process between Sn+ macro-

phages and CD4 and CD8 T cells, liver sections were double-

stained after immune cell transfer with SER4 mAb directed

against the Sn molecule on macrophages and with mAbs against

B7. 1 (CD8O) and B7.2 (CD86), costimulatory molecules in-

volved in antigen presentation. After ADI, a dramatic increase

in the number and staining intensity of Sn+ liver macrophages

was observed. Previously, we found that in ADI-treated groups,

Sn+ macrophages represented about 90% of all Kupffer cells,

whereas in normal livers, their proportion was around 50%.� As

shown in Fig. 5, more Sn+ macrophages expressed B7. 1 mol-

ecules during GvL (A) than during GvH (B). In GvL, the

maximum expression occurred at day 12, when 64% of Sn+

macrophages were B7. 1 + . This level of expression remained

constant until the last time point tested (28 days). In GvH, the

maximum number of Sn + B7. 1 + cells appeared at day 28, with

63% of Sn + macrophages being B7. 1 + . Interestingly, the num-

ber of Sn+ macrophages expressing the B7.2 molecule was very

low in both the GvL and GvH situations. To investigate whether

the increase in the number of host Sn+ macrophages was due to

proliferation or to the recruitment of monocytes/macrophages

from the blood circulation, liver sections were double-stained

with SER and Ki67, a marker for proliferating cells. No double

positivity was found in either the GvL or GvH situations. This

suggests that the increased number is not due to the proliferative

expansion of preexisting cells, but to the recruitment of nonpro-

liferating cells into the organ.

Cluster-associated I Cells Are Proliferating. The ac-

tivation of CD4 and CD8 cells within the clusters was studied by

staining with mAbs against proliferation marker Ki67. Fig. 6

shows the kinetics of total and proliferating CD4 and CD8 cells

in the GvL and GvH situations. In GvL (Fig. 6A), the maximum

number of CD4 cells was reached at day 20. At this time point,

94% of them were Ki67+ . Afterwards, the number of CD4 cells

and, in particular, the number of proliferating CD4 cells de-

creased quickly. The number of infiltrating CD8 T cells was

lower but also showed a peak at day 20, suggesting that both

CD4 and CD8 T cells contribute to the ADI effect in a syner-

gistic manner (4). However, in contrast to the CD4 population,

the majority of CD8 T cells did not express Ki67. In GvH (Fig.

6B), we observed lower numbers of CD4 and CD8 cells than in

GvL, and most of the cells did not proliferate.

Superantigen-reacting V�36 T Cells Form Clusters with

Sn+ Macrophages. To test whether V�36 donor T cells that

become activated by the antitumor immunization of donor mice

and produce Thl cytokines (Table 1) might also infiltrate the

liver upon ADI, double stainings were performed including

mAbs against V�36. Fig. 7 shows the kinetics of V�36 CD4 or

CD8 T cells in the GvL (A) and GvH (B) situations. In tumor-

bearing mice (GvL), the level of V�36 T cell infiltration in-

creased dramatically at day 5 after AD! and then decreased

progressively until day 28. This increase was mostly due to high

proliferative activity, as shown by the Ki67 staining. Most of

these infiltrating and proliferating V�36 T cells were CD4+.

There was an increase in V�36/CD4 T cells from 10% in donor

cells before immune cell transfer to 25% in the host liver. No

increase was observed in the Vf36/CD8 T cells. In non-tumor-

bearing mice, V�36 T cells increased only at day 5, and to a

lesser extent than that in GvL. The same organs were triple-

stained with mAbs against V�36, CD4, CD8, and Sn, and the

amount of double-positive V�36/CD4 or V�36/CD8 T cells lo-

calized in clusters together with Sn+ macrophages was evalu-

ated (Fig. 8). In both the GvL (A) and GvH (B) situation, the

ratio of V�36/CD4:V�36/CD8 double-positive cells was 2: 1 . In

GvL, only about 45% of V�36/CD4 cells formed clusters with



S� .....�:.uj

V � � �

� � :;:!;�.


Fig. 4 Transmission electron

microscopy picture of a section

through a cluster showing tight

interactions between a macro-

phage (K) and two lymphocytes(L) in the liver of a tumor-bearing

mouse 12 days after AD! treat-

ment. Fixed and embedded liver

tissue was ultrathin sectioned and

double-stained with uranylacetateand bead citrate. K, Kupffer cell;L, lymphocyte. Arrows point to

the membrane contact betweenKupifer and lymphocyte cells.Magnification, X 10,000. Bar, 1

p.m.

Sn+ macrophages, whereas all V�36/CD8 cells were within the

clusters. In the GvH situation, the total number of V�36 T

lymphocytes was lower than that in the GvL situation. The

proportion of V�36/CD4 cells in clusters in the GvH situation

was the same as that in the GvL situation, but V�36/CD8 cells in

the GvH situation were found to be associated not only with

Sn+ macrophages, as in the GvL situation, but also as single

cells.

V�6+ Cells Induce Tumor Cytostasis. To further in-

vestigate the possible effect of V�36 T lymphocytes on the tumor

cells in the host liver, we evaluated their tumor cytostatic

activity in vitro. V�36 T cells were isolated by magnetic bead

column fractionation from the spleens of B lO.D2 mice that were

primed or nonprimed with ESb-MP lymphoma cells. The purity

of the isolated V�36 T cells was >85% (fluorescence-activated

cell-sorting staining). ESb-MP cells were cocultured with either

V�36+ or V�36- cells, and the rate of proliferation was meas-

ured by [3H}thymidine incorporation after 48 h. As shown in

Fig. 9, the coculture of ESb-MP cells with V�36+ cells induced

a decrease in tumor cell proliferation (25% [3H]thymidine up-

take when confronted with primed V�36 T cells in comparison to

controls). There was also a dramatic decrease in the rate of

proliferation when tumor cells were cocultured with V�36- T

cells from primed mice. Interestingly, a difference between

V�36+ and V�36- T cells was seen when using nonprimed cells.

The former were significantly more cytostatic than the latter.

DISCUSSION

The data presented in this study demonstrate the formation

of complex cell clusters in the livers of tumor-bearing mice

upon ADI, leading to effective GvL reactivity. The clusters are

composed of macrophages expressing Sn and of CD4 and CD8

T cells. Previous results showed that Sn+ macrophages in-

creased dramatically upon immune cell transfer into tumor-

bearing mice (10). Double staining with anti-Sn mAb and mAb

against donor-type �32 microglobulin showed that this macro-

phage subpopulation was of host origin (21). In the present

study, we found that Sn+ macrophages formed tight clusters

with both CD4 and CD8 donor T cells; thus, these clusters could

be sites of T cell-macrophage and of CD4-CD8 immune T-cell

interactions. Synergistic interaction between immune CD4 and

CD8 donor T cells was previously shown to be a prerequisite for

a successful ADI therapy beading to a complete tumor regression



80.

60.

40.

20.

0

a)

0.�

a-a)

a)C.)

0a

days after AD!

60

50

40

30

20

10

0U

days after AD!


a)

0.�

I.

a)

00

a)

0.�

a..a)

U)

a)

00

- - I I I I I

0 5 10 15 20 25 3

days after ADI

80

B

60�

40.

20

5 10 15 20 25 30

a)

0.�

a-

a)C.)

da

days after AD!

Fig. 5 Kinetics of the accumulation of activated Sn+ macrophages in

livers of GvL (A) and GvH animals (B) under AD!. Frozen tissue

sections were single-stained for Sn+ macrophages (E) or double-stained for Sn+/B7.l + ( � ) or Sn+/B7.2+ ( #{149}) with the corresponding

mAbs. Three to six lobules/liver were analyzed under the microscope.

Data show the means and SDs from two experiments with two to threeanimals/time point. Data points without SD represent the mean valuesfor which the SD was smaller than 1.

and eradication of metastasis (4). The close contact of T cells with

Sn + macrophages, which was demonstrated by electron micros-

copy. makes a functional cooperation of these two types of cells

very likely; for instance, they could interact in tumor antigen

presentation and recognition and in antitumor immune responses.

In support of this assumption, previous in vitro results demon-

strated that Sn + macrophages are able to process exogenous TAA

from metastases and present it via MHC class I! to CD4 (10) and

via MHC class I to CD8 immune donor T lymphocytes.5 During

GvL in our model, Sn + macrophages were found to express

costimulatory molecules such as B7. 1 and, to a lesser extent, B7.2.

It is known that the expression of these two molecules is regulated

differently, and that B7.2 expression tends to precede B7.l expres-

sion during an immune response (22).

Fig. 6 Proliferation of CD4 and CD8 T lymphocytes in the livers of

tumor-bearing (A) and non-tumor-bearing mice (B) under AD! treat-ment. Frozen tissue sections were either single-stained for CD4+ (#{149})orCD8+ (#{149})or double-stained for CD4+/Ki67+ ( � ) or CD8+/Ki67+(�) with the corresponding mAbs. Three to six lobules/liver were

analyzed under the microscope. Data show the means and SDs from twoexperiments with two to three animals/time point. Data points without

SD represent the mean values for which the SD was smaller than 1.

In this study, we also investigated the proliferation status of

the donor T cells that infiltrated the liver, using double staining for

T cell markers and Ki67 as a proliferation-associated nuclear

marker (20). An interesting difference between the liver infiltrates

of CD4 and CD8 T cells was noted: whereas CD4 T cells showed

a high proliferative activity, CD8 T cells had a very low prolifer-

ative activity. Nevertheless, both T cell subpopulations increased in

number during ADI, and this was reflected by the fact that the

number of cells within the clusters varied and were highest in

late-stage GvL. Whether there is a selective recruitment of CD8 T

cells to the metastatic liver remains to be elucidated. Sn+ macro-

phages, which also increased in number during ADI, did not

express Ki67; thus, the possibility of a proliferative expansion from

a liver-derived precursor population is unlikely. The increase in



days after AD!

10

8

6

4

2

0

days after AD!

absolute number, staining intensity, and the percentage of these

cells after AD! may be due to the recruitment of monocytes from

the blood followed by differentiation into Sn+ macrophages in the

environment of the metastatic liver upon immunotherapy. Their

recruitment may be due to the chemotactic factors released from

tumor cells (such as macrophage inflammatory protein lix, which is

expressed in these tumor cells at the mRNA level6) or from

immune cells interacting with the metastatic tumor cells in the liver.

This hypothesis is supported by the finding that the number of Sn+

macrophages in the GvL situation was higher than in the GvH

system.

The molecular basis of the observed cluster formation

needs to be further investigated. One possibility is that the Sn

molecules on the macrophages function as adhesion molecules,

facilitating the anchoring of CD4 and CD8 T cells to their

surface for cell-cell interactions. A similar role of adhesion

molecules, such as leukocyte function-associated antigen I , has

been reported for DCs interacting with resting B cells as a

prerequisite for antibody synthesis (23) and in the homotypic

cluster formation of human adult T-cell leukemia as an initial

and essential step for cytokine production, proliferation of hu-

man adult T-cell leukemia, and hypercalcemia in vivo (24).

Clustering of DCs, Th cells, and histocompatible B cells also

occurs during primary responses in vitro (25). Other immune

processes, such as the generation of specific CTLs, take place in

6 K. Jurianz, P. von Hoegen. and V. Schirrmacher. Immunological and

molecular characterization of an aggressive murine lymphoma: modu-

lation in vitro and in vivo, manuscript in preparation.


a)

0.�

a-

a)

U)

a)C.)

da

a)

0

a-

a)C.)

da

Fig. 7 Characterization of the V�36 T-celb population in the livers of

GvL (A) and GvH (B) animals under ADI therapy. Frozen tissue sections

were stained for V�36, CD4, CD8, and Ki67 with the corresponding

mAbs. The figure shows the total number of V�36+ T cells ([1) andV�36+/Ki67+ (#{149}),V�36+/CD4+ (i), and V�36+/CD8+ (0) double-

positive cells. Three to six lobules/liver were analyzed under the mi-

croscope. Data show the means and SDs from two experiments with two

to three animals/time point. Data points without SD represent the meanvalues for which the SD was smaller than 1.

a)

0

a-

a)C.)

da

a)

0

a-

..0

a)U

0a

Fig. 8 Cluster formations of V�36+/CD4+ and V�36+/CD8+ T lym-

phocytes with Sn+ macrophages in the livers of tumor-bearing (A) andnon-tumor-bearing (B) mice under ADI treatment. Frozen tissue sec-

tions were triple-stained for V�36, Sn, and CD4 or CD8 with the

corresponding mAbs. The figure shows the total numbers of V�36+ (I).V�36+/CD4+ (El), and V�36+/CD8+ (D) T lymphocytes and the num-ber of V�36+/CD4+ (�) and V�36+/CD8+ (�) T cells localized in

clusters with Sn+ macrophages. Three to six lobules/liver were ana-

lyzed under the microscope. Data show the means and SDs from twoexperiments with two to three animals/time point. Data points without

SD represent the mean values for which the SD was smaller than 1.



100.


Fig. 9 Influence of V�36 T cells on the proliferation of ESb-MP tumorcells in vitro. Tumor cells were cultured for 48 h alone (U), in combi-

nation with nonprimed (�) and ESb-MP-primed (�) BlO.D2-derived

V�36- T cells or nonprimed (�) and primed (fl) V�6+ T lymphocytes,respectively. The proliferation rate of ESb-MP tumor cells was meas-

ured in three independent experiments using the l3Hlthymidine assay.

Data are expressed as a percentage of the proliferation rate of tumor

cells cultured alone (control). Bar. means ± SD.

cell clusters composed of cytotoxic and several Th cell subsets

collaborating on the surface of APCs (26). In this process, T-cell

subsets may require simultaneous antigen recognition and mem-

brane interactions with APCs. Such a mechanism is likely to

occur in our model, because we observed intensive membrane-

membrane contacts between APCs and T cells by electron

microscopy. An attractive possibility is the recently published

“dynamic model” (27) that proposes that the three cells types,

CD4 T cells, CD8 T cells, and APCs such as DCs, need not

interact simultaneously, but sequentially. The helper cell can

first engage and condition the DC, which then becomes licensed

to stimulate a T killer cell. The identity of other molecules in

APCTF-cell adhesion remains to be elucidated. If APCfF-cell

adhesion uses mechanisms similar to those involved in leuko-

cyte/endothelium adhesion, we would predict the involvement

of integrins, chemokines, and their receptors.

The effectiveness of the described ADI was evaluated by

staining the metastatic livers after immune cell transfer with

proliferation marker Ki67. Tumor proliferation is usually deter-

mined by the number of tumor cells found in mitosis. The high

number of clusters of proliferating cells in the liver early after

cell transfer (day 2) indicated the presence of metastatic foci

before donor immune cells reached the liver (day 5: Ref. 20).

The number of tumor foci decreased with time of therapy, and

they were absent from organs at a later stage of treatment (Fig.

I ). The complete eradication of late-stage macroscopic metas-

tases by this ADI protocol was also demonstrated noninvasively

in intact animals by ‘H nuclear magnetic resonance microim-

aging with respiratory triggering (28). In addition, noninvasive

in tis’o � ‘ P nuclear magnetic resonance spectroscopy revealed

an early significant change due to ADI in tumor tissue pH by

day 5-6 after treatment (29). Whether the observed disappear-

ance of immune donor spleen cells from the liver at the later

time points is due to apoptosis induced by host cells involved in

host-versus-graft reactivity is under investigation. Host-versus-

graft reactivity is expected to recover in 5-Gy-treated animals in

about 3 weeks. Preliminary data indicate that the production of

NO increased dramatically at day 20 after AD!, coinciding with

the disappearance of immune cells.4 Previous data show that NO

induces apoptosis in both murine and human tumor cells (30,

3 1 ). NO could thus be involved in the apoptosis of immune

donor cells after therapy as a mechanism of their clearance from

the liver.

In previous studies, we reported on the extraordinary im-

munoresistance of B l0.D2 mice to highly metastatic tumor lines

such as ESb derived from the mouse strain DBAJ2 (32, 33). We

have recently found that when typed for MMTV proviruses in

their genome, BbO.D2 and other resistant recombinant inbred

mouse strains showed a selective boss of a long terminal repeat

hybridization band that corresponded to Mtv-7. An open reading

frame in the long terminal repeat of Mtv-7 codes for vSAG7

(Mls�’), an autoantigen of DBA/2 that causes the deletion of

superantigen-reactive T-lymphocytes such as V�36+ cells dur-

ing thymus maturation (34, 35). Suggestive evidence for a link

between tumor resistance and the absence of endogenous vSAG

was given by a back-cross segregation analysis as well by the

reintroduction of the superantigen into the resistant line and the

reappearance of tumor susceptibility (5). ESb tumor cells, as

well as the syngeneic mouse strain DBA/2, express proviral

Mtv-7 at the mRNA level.4 It is possible that after the injection

of ESb tumor cells into B l0.D2 tumor-resistant mice that do not

express the superantigen Mtv-7, V�36 superantigen-reactive T

cells become activated and, after an adoptive transfer into ESb

tumor-bearing DBA/2 mice, infiltrate ESb liver metastasis and

recognize the superantigen. Other groups have reported that

human V�36 T cells recognize TAAs in ovarian cancer (36),

gastric carcinomas (37), adrenal carcinomas (38), and neo-

plasms of the central nervous system (39). In our GvL mouse

model, it may be important to find out how superantigen-

reactive V�36 T cells contribute to the therapy effect. Therefore,

we double-stained metastatic livers with antibodies against V�36,

CD4, CD8, and Ki67 at different time points after therapy.

During GvL, V�36 T cells possessed a high proliferation rate and

were found more in the CD4 T-cell population than in the CD8

T-cell population. In GvH, V�36 T cells were almost absent. This

supports the hypothesis of a reactivity of these cells against the

tumor-associated superantigen. V�36 T cells were also present in

clusters in contact with Sn+ macrophages. Superantigens are

known to bind to MHC class II molecules and to certain V�3

T-cell receptor chains, thus resulting in the cross-linking of both

molecules and the subsequent activation of the T cell (40). In

our model, the interaction of the superantigen and the MHC

class I! molecules in the Sn+ macrophages would need the

cleavage of the superantigen from the surface of the tumor cells

and then the transfer of the cleaved form to the MHC class I!

molecules on the Sn+ macrophages. vSAGs contain furin-like

cleavage sites and are naturally cleaved. In particular, vSAG7 is

synthesized as a Mr 45,000 transmembrane glycoprotein precur-

sor but is proteolytically processed to yield a Mr 18,500 surface

protein that is the functional form of the superantigen (41).

Second, the interaction of this functional form of the superan-

tigen with the Sn macrophages might happen by paracrine

transfer as described previously ( I 1 ) in a process that requires

not cell-cell contact but cellular proximity. Additional experi-




ments are required in our model to find out the mechanism of

recognition, binding, processing, and presentation of the tumor-

associated Mtv-7 superantigen.

To test whether V�36 T cells could have a direct effect on

tumor cells, we performed an in vitro assay in which immu-

nobead purified V�36 T cells from primed and nonprimed

mice were cocultivated with ESb-MP tumor cells. The data

presented show a direct cytostatic effect of V�36 T cells on

ESb-MP tumor cells. Interestingly, the effect was very sim-

ilar in V�36 T cells from primed and nonprimed mice. In

contrast, only primed V�36- cells produced a strong antitu-

mor cytostatic effect. One may speculate that other cells

besides the V�36 T cells are also involved in the observed

therapeutic effect and need to be primed before the therapy to

recognize either minor histocompatibility antigens or the

TAA. This would suggest that in vivo, more than one T-celb-

mediated effector mechanism (SAG-reactive and TAA-reac-

tive cells, CD4 and CD8 T cells) needs to be operative, and

that cooperation between T cells must be established to

achieve the observed complete tumor regression of late me-

tastases. V�36- T cells recognizing minor histocompatibibity

antigens and TAAs would need to be primed before transfer,

but V�36+ cells recognizing the superantigen directly would

not require priming. We also provide evidence that donor

V�36 T cells are activated after tumor cell injection and

produce high amounts of IL-2 and IFN--y just before cell

transfer. This activation and cytokine production may be

particularly important for other subpopulations of donor T

lymphocytes. We suggest that V�36 donor T cells are able to

mediate antitumor activity after recognition of the Mtv-7

superantigen on the surface of ESb tumor cells and in sites of

liver metastases during AD! in this GvL model.

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