Lack of T-Cell-mediated Recognition of the Fusion Region of the … · Vol. 2. 593-600. Marc/i 1996 Clinical Cancer Research 593 Lack of T-Cell-mediated Recognition of the Fusion

Vol. 2. 593-600. Marc/i 1996 Clinical Cancer Research 593

Lack of T-Cell-mediated Recognition of the Fusion Region of the

pmLIRAR-a Hybrid Protein by Lymphocytes of Acute Promyelocytic

Leukemia Patients’

Said Dermime, Carla Bertazzoli,

Edoardo Marchesi, Fernando Ravagnani,

Kurt Blaser, Gian Marco Corneo,

Enrico Pogliani, Giorgio Parmiani, and

Carlo Gambacorti-Passerini2

Division of Experimental Oncology D and Blood Bank, Istituto

Nazionale Tumori, Via Venezian 1, 20133 Milan. Italy [S. D.. C. B..

E. M.. F. R., 0. P.. C. G-P.l: Swiss Institute of Allergy and Asthma

Research. Davos, Switzerland [K. B.]: and Section of Hematology.

University of Milan, S. Gerardo Hospital, Monza, Italy [G. M. C.. E. P.1

ABSTRACT

In previous studies, it was shown that the fusion regionof the pmIIRAR-a protein, expressed by acute promyelo-

cytic leukemia (APL) cells, can be specifically recognized in

vitro by donor (D. E.) CD4 T cells in a HLA class II DR11-restricted fashion. We present here the results on the rec-

ognition of several pm1IRAR-a peptides by APL patients

expressing HLA DR1 1. The in vitro immunization of peniph-

eral blood lymphocytes from four patients in remission

(S. R., F. R., M. M., P. G.) with BCR1/25, a 25-men pml/

RAR-a, did not elicit either a polyclonal or a clonal immuneresponse specific to the peptide. We then generated new

donor anti-pmlIRAR-a CD4� 1-cell clones. These cloneswere tested for their recognition of BCR1/25. One clone

(C3/5, CD3�, CD4�, CD8) was selected for further analy-sis. Clone C3/5 showed specific proliferation, cytotoxicity,and cytokine (tumor necrosis factor a, granulocyte-mac-rophage colony-stimulating factor) production when chal-

lenged with autologous lymphoblastic cell lines pulsed with

peptide BCR1/25. C3/5 cells developed specific proliferation

and cytotoxicity when challenged with peptide-pulsed lym-phoblastic cell lines and peripheral blood lymphocytes from

the four DR1 1 � APL patients. APL blasts, available onlyfrom patients F. R. and P. G., were not lysed by C3/5 andwere unable to present peptide BCR1/25. Incubation of APL

cells with IFN-�j failed to induce HLA class II molecules and

recognition by the C3/5 clone. Since APL cells do not express

HLA class II molecules, we tested in two donors (D. E. and

C. H. R.) and in patients S. R. and P. G. whether the use of9-mer peptides (BCR1/9) would generate a CD8IHLA class

I-restricted response. No peptide-specific 1-cell line or clone

Received 6/15/95; revised 10/18/95: accepted 1 1/28/95.I This work was supported in part by the Italian Association tir Cancer

Research. the Italy-United States Program on Therapy ofTumors (Grant

531 23.12.87). and a donation from CEVA SpA, Monza.

2 To whom requests for reprints should be addressed. Phone: 39.2.239-0207: Fax: 39.2.236-2692.

could be generated from both donors and patients. These

findings are discussed in relation to possible therapeutic

approaches to the immunotherapy of APL.

INTRODUCTION

The pml/RAR-cs fusion protein, specifically expressed in

APL3 ( 1-4) cells. creates a new tumor-specific amino acid

sequence. It has been shown that a 25-men peptide (BCRI/25),

encompassing the fusion region, contains an antigenic site (ab-

sent in the normal parent molecules) recognizable by CD4�

T-cell clones of a healthy donor (D. E.) in an HLA DR1 I-

restricted fashion on presentation by autologous APCs (5. 6).

Antipeptide CD4 clones also recognize pml/RAR-a-transfected

LCLs (6).

The possible recognition of the pml/RAR-a protein by 1

lymphocytes of APL patients would open the possibility of

directing the patient’s immune system against the leukemic cells

through the recognition of a tumor-specific/transformation-re-

lated molecule (5). Although binding motifs for most DR mol-

ecules were not known at the time of the study, our previous

data (6) indicated that DR1 1 functions as a restriction element in

this case. For this reason, we focused on the analysis of DR 1 1 �

of DRI 1 APL patients.

In the present work, we studied the in vitro immune re-

sponse of peripheral blood lymphocytes from four HLA DR1 I

APL patients to several pml/RAR-a peptides.

PATIENTS AND METHODS

Patients

Four APL patients were studied after informed consent was

obtained. Three patients (F. R., S. R., and P. G.) were in first

remission, and one patient (M. M.) was in second remission.

Two patients (S. R. and M. M.) underwent autologous bone

marrow transplantation: the conditioning regimen included

high-dose chemotherapy but not total-body irradiation. At the

time of the study, patients had not received treatment for at least

6 months. and their peripheral counts were within normal

ranges.

I The abbreviations used are: APL. acute promyelocytic leukemia: LCL,lymphoblastoid cell line; APC. antigen-presenting cell; PBMC. periph-

eral blood mononuclear cell: LAK. lymphokine-activated killer: IL.

interleukin; HPLC, high-performance liquid chromatography: HS. hu-

man serum; dThd, thymidine; PBL. peripheral blood lymphocyte:

TNF-a. tumor necrosis factor a; GM-CSF, granulocyte-macrophage

colony-stimulating factor; MAb, monoclonal antibody; PHA, phytohe-

magglutinin: TT, tetanus toxoid; SI. stimulation index; TCR. T-cell

receptor.

on April 8, 2021. © 1996 American Association for Cancer Research.clincancerres.aacrjournals.org Downloaded from

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594 Immune Recognition of the pml/RAR-a Protein

PBMCs

Peripheral blood was obtained from two healthy donors

(D. E. and C. H. R.) and four APL patients in remission (S. R.,

F. R., M. M., and P. G.) after informed consent. PBMCs were

isolated by centnifugation on Ficoll gradients. The HLA typing

was as follows: DE (A2,23, B49,35, DR1 1,13), CHR (A2,l I,

B35,60, DR1,4), SR (Al,24. B51, DR8,ll), FR (A23, B49,5l,

DR4,l 1), MM (A23,l0, B27,49, DRIO,l I), and PG (Al,2, Bl5,

DRI,l I).

Cell Lines

Lymphoblastoid cell lines (DE.LCL, SR.LCL, FR.LCL,

MM.LCL, PG.LCL, and CHR.LCL) were prepared from PBMCs

as previously described (6). LAK cell lines were prepared by

culturing PBMCs with recombinant IL-2 (l0� units/ml; Euro-

Cetus By, Amsterdam, the Netherlands). The Daudi cell line

was cultured in RPMI 1640 (Biowhittaker, Walkersville, MD)

plus 10% FCS (Biological Industries, Kibbutz Beth Haemek,

Israel). FR.APL cells were obtained from bone marrow or

peripheral blood of an APL patient (F. R.) and frozen in liquid

nitrogen. They were thawed and cultured for I 8 to 24 h before

use in proliferation or cytotoxicity tests.

Peptides

The following peptides were used: BCRI/25, a 25-men

(NSNHVASGAGEAAIETQSSSSEEIV) peptide, and a pool of

nine different 9-men peptides (VASGAGEAA, ASGAGEAAI,

SGAGEAAIE, GAGEAAIET, AGEAAIETQ, GEAAIETQS,

EAAIETQSS, AAIETQSSS, and AIETQSSSS) collectively in-

dicated as BCRI/9. They encompass the BCR1-type fusion

region of the pml/RAR-a protein (4). The peptides were syn-

thesized by the University of Wisconsin Biotechnology Center

(Madison, WI) or American Peptide Company (Sunnyvale, CA)

and purified using high-performance liquid chromatography to a

minimum purity of 95%.

Activation of Lymphocytes

The protocol described by Chen et a!. (7) was followed. In

brief, 50 x 106 fresh PBMCs (l0� for patient P. G. and donor

C. H. R.) were incubated in a humidified atmosphere at 37#{176}Cin

5% CO, with 20 p.M corresponding peptides at 5 X 106 cells/mI

serum-free RPMI 1640 medium for I h. Fresh autologous PB-

MCs (50 X 106 cells in 10 ml RPM! 1640 medium + 10% HS)

were added to each APC culture in culture flasks (75 cm2). The

flasks were incubated upright for 7 days. Primed lymphocytes

were then harvested and restimulated with irradiated (3000 rad)

autologous PBMC-pulsed peptides (20 �J.M) at a lymphocyte:

APC ratio of I :3. The next day, IL-2 (10 units/ml) was added,

and the percentage of HS was increased to 10%. Five days later,

one half of the medium from each culture was replaced with

RPMI 1640 + 10% HS + 10 units/ml IL-2. The derived T-cell

lines were left in culture for 7 days before cloning on day 21

poststimulation. They were maintained in culture by periodic

stimulation (every 7-10 days) using irradiated (8000 rad) auto-

logous LCL-pulsed peptide (20 �.LM) and IL-2 (10 units/mi). To

test for proliferative activity of the derived T-cell lines, cells

were washed and placed in U-bottomed 96-well plates (5 X l0�

cells/well). They were stimulated with irradiated (8000 nad)

autologous LCLs (l0� cells/well) in the presence or absence of

the appropriate peptides. LCL cells were washed three times in

serum-free RPMI 1640 medium, incubated with 20 �LM peptides

as indicated above, and then irradiated. After 48 h of culture, the

plates were labeled with [3H]dThd, further cultured for 18 h,

harvested, and counted in a beta counter.

Generation of Lymphocyte Clones

Primed 1-cell lines were cloned on day 21 by limiting

dilution (20, 10, 5, 2, and 1 cell/well/200 pA RPM! 1640 + 10%HS) in 96-well U-bottomed plates. Autologous LCLs, PBLs,

and allogeneic PBLs (25 X l0� cells/well) were used as a feeder

layer. Autologous PBLs and LCLs were incubated separately in

serum-free RPM! 1640 medium (5 X 106 cells/ml) with 20 �iM

of the same mixture of peptides, used to prime lymphocytes (see

above), for 30 mm at 37#{176}Cbefore irradiation (3000 rad for PBLs

and 8000 rad for LCL cells). X-irradiated (3000 rad) allogeneic

PBLs and IL-2 (50 units/mI) were also added to the wells. The

plates were incubated for 14 days; 100 jil fresh medium and 50

units/ml IL-2 were replaced in each well every 3 days. Wells

with positive signs of growth were selected for expansion (see

‘ ‘Results’ ‘ ). The probability of clonality for each well contain-

ing growth was calculated as described previously (6). Growing

clones were transferred to 96-well flat-bottomed plates, and each

clone was stimulated with 25 X l0� irradiated (8000 rad) autol-

ogous LCLs pulsed with the appropriate peptides (20 jiM) and

25 X l0� (3000 rad) irradiated allogeneic PBLs. !L-2 (50

units/mI) was added to the wells after I day. This activation

cycle was repeated after 7-10 days of culture at lymphocyte:

autologous LCL:allogeneic PBL ratios of I : 1 : I . unless other-

wise indicated.

Screening of Clones

The following assays were carried out.

Proliferation Assay. 1-cell clones were first screened

for proliferative activity 28 days after cloning. Each well was

washed off to remove IL-2 and split into three. The first one was

expanded for further culture. The second well was stimulated

with irradiated (8000 rad) autologous LCLs ( l0� cells/well)

pulsed with the appropriate peptides (20 �1M). The third well was

incubated with irradiated LCLs without peptides. Cells were

cultured for 48 h, incubated for an additional I 8 h with

[3HJdThd (1 p�Ci/well), and harvested. In subsequent expeni-

ments, the incubation time was reduced to 24 h (see “Results”).

Cytotoxic Assay. T-cell clones were first screened for

cytotoxic activity 6 weeks after cloning: one third of the cell

suspension from each clone was admixed with 5tCr-labeled

autologous LCLs (10� cell/well) as a control, and the other third

was incubated with LCLs pulsed with peptide (20 pM). The

assay was performed as previously described (6). The incuba-

tion time was 4-6 h. Spontaneous release never exceeded 15%.

Anti-TNF-a monoclonal antibody (Farmitalia, Milan, Italy) and

TNF-a (EuroCetus BV) were used in some assays (see � ‘Results”).

Cytokine Production and Detection Assays

Cells from clone C3/5 (3 X 106 cells) were cultured in

48-well plates (1.5 X 106 cells/mi) with irradiated (8000 rad)

autologous DE.LCL (3 X 106 cells) in the presence on absence



SR/9 SR/25 IDE/9/25

Po1�cloma1 T cell lines

Clinical Cancer Research 595

of 20 J.LM peptide (25-men). C3/5 cells (3 X 10#{176})were also

cultured in 2 ml RPMI 1640 + 10% HS in the absence of LCLs.

The supernatant from each culture (2 ml) was collected by

centnifugation after I and 2 days. immediately frozen in liquid

nitrogen. and stored at -80#{176}C until used. The cytokines assayed

in the present study were: IL-2, IL-3, IL-4, IL-6, IFN-y, TNF-a,

and GM-CSF. IL-2 activity was measured by [3HJdThd uptake

of IL-2-dependent cytotoxic T lymphocyte line cells as previ-

ously described (Ref. 8. 1 unit biological activity was referred as

half-maximal proliferation of cytotoxic T lymphocyte line

cells). In brief, serial dilutions of IL-2 or supernatant from each

culture were incubated in 200 �il for 24 h, followed by an

overnight pulse of I p.Ci/well of I3HldThd. IL-3 was assayed by

using a Quantikine Human IL-3 Immunoassay (Research and

Diagnostic Systems, Minneapolis, MN) with a detection limit of

30 pg/mi. IL-4 and IFN--y were measured using the sandwich

ELISA as described in detail elsewhere (9). The mouse MAb

8F I 2 ( 10) and biotinylated 3H4, generously provided by C. H.

Heusser (Ciba-Geigy Ltd.. Basel. Switzerland), were used for

IL-4, and MAbs 43-I I and biotinylated 45-15. kindly supplied

by S. Alkan (Ciba-Geigy Ltd.. were used for IFN--y. The

sensitivities of the lL-4 and IFN--y ELISA were 30 and 40

pg/nil. respectively. IL-6 and TNF-cs were analyzed using kits

from Chromogenix AB (Molndal. Sweden), with a detection

limit of 4 pg/mI as given by the manufacturer. The human

GM-CSF was assayed using Innotest human GM-CSF from

Innogenetics NV (Antwerp, Belgium) with a detection limit of

8 pg/ml.

PHA and TI Assays

PBLs were simultaneously thawed, washed, and seeded at

l0� cells/well. PHA (Murex, Temple Hile. England) was added

at I �ig/rnl, and proliferation was assessed as [3H]dThd uptake

(6-h incubation) after 72 h. For proliferation to Ti’ ( 10 p.g/ml:

Connaugh Lab), 2 x l0� PBLs/well were cultured for 6 days

and labeled with l3HIdThd tc�r the last 18 h of culture.

Immunofluorescence Assay

This assay was performed as previously described ( I I).

The MAbs used were in the form of diluted ascites: anti-CD3.

anti-CD4, anti-CD8, anti-TCR-ct�3, anti-TCR-y�, anti-HLA

class I (W6/32). and anti-HLA class II (Dl-12). Samples were

analyzed using flow cytometry (FACs IV: Becton Dickinson,

Sunnyvale. CA) with a logarithmic signal amplification.

Screening for HLA-binding Motifs

A list including the binding motifs for 35 HLA class I

specifIcities was kindly supplied by Dr. H. G. Rammensee

(Heidelberg. Germany) and includes the following specificities:

Al. 0201/5. 3. 11. 24. 31. 33, 68: B7, 8, 2702/5, 3501/3, 3701,

3901/2. 40. 4402/3. 5101/2/3, 5201, 53, 58. 60, 61, 62, 78;

CwO3Ol. 0401, 0602, 0702.

HLA Stabilization Assay

The ability of peptides to bind HLA molecules was as-

sessed by the peptide-mediated increase in the amount of HLA

expression on the membrane of the 12 ( I 2). L72 1 .22l( 1 3), and

ST-EMO ( 14) cell lines. Cw6 and Cw0702 transfectants of T2

20000

17500

15000

�- 12500

U

C10

� 7500

5000

2500

0

Fig. I Proliferative response of three polyclonal T-cell lines ISRI9,

SR125 ((mm an APL patient in remission), and DE/9/25 (from a healthy

donor)l against BCRI/9. BCR1/25, or BCRI/9 + BCR1/25 peptides,respectively. Each T-celI line (5 X l0� cells/well) was cultured for 48 h

with irradiated (80(X) rad) autologous EBV-transformed LCLs (l0�

cells/well) in the presence (� or absence (m) of the corresponding

peptide (�, lymphocytes cultured in RPMI 1640 + 10% HS in the

absence of both LCLs and peptides. They were incubated ftr an

additional 18 h with i�HidThd and harvested. LCLs were pulsed with

peptides in serum-free medium for 30 mm before irradiation. cpm

values represent the mean of six replicates. Bars. SD.

and L721.22l were also used (kindly supplied by Dr. D. Schen-

del, Munich Germany). Cells were washed in serum-free RPMI

1640 and incubated with 20-100 pg/ml peptide for 18 h at

37#{176}C.Expression of HLA class I molecules was evaluated by

indirect immunofluorescence with W6/32 MAb or with mono-

specific MAbs GAP-A3, 4E (anti-B/C), or BB7.2 (anti-A2).

Positivity was defined as an increase by at least 50% in the

median channel of fluorescence.

RESULTS

Polyclonal 1-Cell Lines. The results for the proliferative

activity of 1-cell lines, obtained from an APL patient in nemis-

sion (S. R.) and a healthy donor (D. E.), are presented in Fig. I.

SR/9 and SR/25 were obtained by culture in the presence of

BCRI/9 or BCRI/25; DE/9/25 was derived from cultures in the

presence ofboth BCR1/9 and BCRI/25 peptides. SR 1-cell lines

did not show a specific proliferative activity against autologous

peptide-pulsed LCLs. A low but reproducible SI (SI cpm test

well/cpm control well) of 2.2 was obtained for the DE/9/25

T-cell line. These lines consisted of a mixture of CD4 and

CD8-a/�3 lymphocytes. No specific cytotoxic activity against

peptide-pulsed LCLs was obtained in these cell lines (data not

shown).

1-Cell Clones. The 1-cell lines SR/9, SRJ25, and DE/

9/25 were cloned by limiting dilution. Two hundred fifteen

clones (with a clonal probability of >90%) were obtained; 52,

41, and 122 clones were generated from SR/9, SR/25, and



100

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596 Immune Recognition of the pmlIRAR-cs Protein

Fig. 2 Cytotoxic activity of clone C3/5 (E. effector cells) against

autologous DE.LCL (T, target cells) in the presence of BCR1/9 (B) and

BCRI/25 (C) or absence (A) of peptide. LCLs at 5 X 106 cells/ml were

incubated with 51Cr for 90 mm with or without peptides (20 p.M). After

washing. the peptides were readded to the target (10� cells/well) at the

same concentration.

DE/9/25. respectively. Among the 52 SR/9 and the 41 SR/25

clones tested, no specific cytotoxic or proliferative activity was

obtained. Proliferation against LCLs pulsed with control pep-

tides or with PBS was observed in several clones, suggesting

autoreactivity or anti-EBV reactivity (data not shown). Among

the 122 clones obtained from the DE/9/25 T-ceil line, 59 were

selected according to their specific proliferation (SI >2) or

cytotoxicity (specific lysis >10%). Fifty-five clones showed

specific proliferation to the peptide pool (SI ranged from 2 to

16.47) with fourciones showing specific lysis also [B9/5 (17%),

C3/l (20%), C3/5 ( 19%), and C4/l (13%)1. An additional four

clones showed specific lysis only [A4/5 (19%), E2/l (10.5%),

E3/l (23%), and F2/l ( 15%)]. The phenotype of these clones

was uniformly CD3�, CD4�, CD8, TCR-a/�, and TCR-

-y/�l� . One clone (C3/5), which showed the highest and the most

stable results, was selected for further analysis. In subsequent

experiments the proliferative and cytotoxic activity of the C3/5

clone against BCRI/25 and BCR1/9 was evaluated. BCR1/9 did

not induce specific proliferation (data not shown), indicating

that BCR1/25 was the peptide recognized in the peptide mixture

initially used for stimulation. The results for the cytotoxic ac-

tivity of C3/5 against autologous LCLs pulsed separately with

BCR1/25 and BCR1/9 are shown in Fig. 2.

T-cell clones were also produced, with the same stimula-

tion protocol and using combined or separate stimulation with

BCRI/25 and BCRI/9, from three additional DR1 I � APL pa-

tients (F. R., M. M., and P. G.) and with BCR 1/9 from donors

C. H. R.. L. R., and B. C. A total of 1015 clones (155 from

patient F. R., 106 from patient M. M., 251 from patient P. G.,

Fig. 3 Specific cytokine production by the C3/5 clone. Cells ( 1 .5 X

l06/ml) were cultured with irradiated (8000 rad) autologous DE.LCL

(1.5 X 106 cells/mI) in the presence (#{149}and V) or absence (0 and 7) of

the BCR1/25 peptide (20 pM). Supernatants were harvested on days 1

and 2 and assayed for the presence of TNF-a (0 and #{149})and GM-CSF

(V and Y).

198 from donor C. H. R., 227 from donor L. R., and 188 from

donor B. C.) was obtained and screened. No specific prolifera-

tive or cytotoxic activity was observed, while a specific re-

sponse was obtained from donors’ PBLs (all being HLA-A2�)

in response to the influenza peptide GILGFVFTL (data not

shown).

Specific Cytokine Production by the C3/5 Clone in

Response to Autologous Peptide-pulsed LCLs. To assess

cytokine production, cells from the C3/5 clone were stimulated

with autologous DE.LCL in the presence or absence of the

BCR1/25 peptide (20 p.M), and cytokine production was mea-

sured after 1 and 2 days of culture. !L-2, IL-3, !L-4, IL-6, IFN-�,

TNF-a, and GM-CSF were assayed. IL-2, IL-3, IL-4, IL-6, and

IFN--y levels were undetectable or below the detection limit of

the assay used (data not shown). C3/5 cells cultured with autol-

ogous DE.LCL-pulsed peptide for I day secreted significant

amounts ofTNF-a (933, 1 189 pg/ml) and GM-CSF (3395, 3301

pg/ml) in two separate experiments compared with those stim-

ulated with DE.LCL in the absence of peptide (26 pg/mi or

below the detection limit for TNF-a and 184, 1 26 pg/ml for

GM-CSF). TNF-a and GM-CSF production peaked at day 1 and

remained stable at day 2 (Fig. 3). To test whether TNF-ct

produced by the C3/5 clone could be involved in its cytolytic

activity, TNF-a and anti-TNF-a MAb were used. The addition

of anti-TNF-a at different concentrations 0. 1, 1 , and 10 p.g/ml

to the target cells DE.LCL in the presence or absence of the

peptide, did not inhibit the cytolytic activity exerted by the C3/5

clone (Fig. 4). The antibody was instead active in inhibiting the

cytotoxic activity of TNF-cs against the TNF-a-sensitive WEHI

164 cell line (data not shown). TNF-a, at 1-1000 units/ml, had

no cytolytic activity on DE.LCL cells when added for the same



60

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Fig. 4 Effect of MAb directed against TNF-a produced by the C3/5clone on the cytotoxic activity. Cells from the C3/5 clone were chal-

lenged with autologous DE.LCL in the absence of peptide (0) and in the

presence of 20 �iM BCRL/25 peptide (#{149}).Anti-TNF-a at 0. 1 �.g/ml (V),1 �ig/m1 (Y), and 10 p.g/ml (�I1) was mixed with LCL-pulsed peptide

(BCR1/25) before the effector cells were added.

duration of the cytotoxic assay (data not shown). The results

indicate that cytotoxic activity and TNF-a release probably

represent two separate functions in clone C3/5, although both

are specifically induced by BCR1/25.

Reactivity of C3/5 toward HLA-DR11 Allogeneic APLs,

LCLs, and PBLs. Five aliogeneic LCLs [SR. FR, MM, PG

(sharing DR1 1 with DE and obtained from APL patients) and

CHR (DR1 1 �)} were subjected to a cytotoxic test in the pres-

ence or absence of BCR1/25 (20 �.LM) at different E:T ratios. The

results are shown in Fig. 5. The C3/5 clone specifically lysed

DR1 1-matched SR. FR, and MM [and PG (data not shown)j

LCLs pulsed with BCR1/25, as well as the autologous DE.LCL.

CHR cells failed to present the peptide to the C3/5 clone. The

ability of the C3/5 clone to proliferate to the autologous

DE.LCL and HLA-DR1 1-matched SR. FR, MM, and PG.LCL

pulsed with different peptide concentrations was also investi-

gated. A preliminary experiment was carried out to determine

the optimal stimulation period giving the highest proliferative

activity of clone C3/5 at this stage of culture. Cells were

cultured for 24, 48, and 72 h with autologous irradiated (8000

rad) DE.LCL in the presence (2 and 20 p.M) or absence of the

BCR!/25 peptide, incubated for an additional 18 h with

[3HJdThd, and harvested. As shown in Fig. 6A, the 24 h- time

point gave the highest cpm count (P < 0.05 using the t test

analysis). The proliferative activity of the C3/5 clone against

autologous and allogeneic LCL-pulsed peptide (0, 5, 20, and 50

ELM) was subsequently studied at the 24-h time point (Fig. 6B).

All DR1 1 � peptide-pulsed LCLs stimulated the C3/5 clone in a

dose-dependent fashion (PG.PBL induced an uptake of 38,500

i.e

0�

3:1 6:1 12:1 24:1

E?f’ector: Target ratio

Fig. 5 Cytotoxic activity of clone C3/5 against autologous DE.LCL in

the absence (0) or presence (5) of peptide, and allogeneic SR.LCL (V

and V), FR.LCL (LI and U), MM.LCL (A and A), and CHR.LCL ( c�and #{149}) cells in the presence (#{149},U, A, and #{149}) or absence (0, LI. A, and

� ) of 20 pM BCR1/25.

± 2,300 cpm). Although some discrepancies between prolifer-

ation and cytotoxicity were observed (SR and MM.LCL induced

a good proliferative response but produced low levels of cyto-

toxicity, while FR.LCL induced little proliferation but was very

efficiently lysed by C3/5), all four APL patients’ LCLs were

able to present peptide BCR1/25 to C3/5. The ability of LCLs

derived from APL patients to present peptide BCR1/25 to C3/5

suggests that the failure in raising anti-pm1IRAR-ct clones from

the four APL patients was probably not due to a defect in

antigen presentation capability. To further investigate this point,

this experiment was replicated using patients’ PBLs (instead of

LCLs) as APCs. The results are presented in Table I , and

confirm that the antigen presentation capability in the APL

patients studied was grossly intact.

Since the C3/5 clone was specifically able to lyse FR.LCL

cells pulsed with the BCRI/25 peptide in a HLA class II (DR1 I)

restriction fashion, we further tested whether this clone could

exert a cytotoxic activity against FR.APL cells. Because APL

cells are usually DR , FR.blasts (�85% leukemic cells) were

also cultured with IFN--y (a cytokine known to induce or en-

hance the expression of HLA class II molecules) before being

used. The results are present in Fig. 7. C3/5 cells did not lyse

FR.APL blasts and the culture of FR.APL cells with IFN-�y (1 0�

units/mi) for 48 h before the experiment did not enhance the

cytolytic activity. This was possibly due to the failure of IFN-’-y

to induce HLA class II molecules on FR.APL blasts as assayed

by immunofluonescence (data not shown). FR.APL cells were

also unable to present peptide BCR1/25 even after IFN--y pre-

incubation. To test whether the interaction of C3/5 cells with

peptide-pulsed FR.LCL could kill the APL blasts by a bystander




Time in culture (hours)

80

70

U).� 60

p-I �#{216}0

�4#{216}.

>5,

10

0

22500

20000

17500

E 150000.0 12500

� 10000wE

5000

2500

0

45000

40000

36800

E0.I) 25000

� 2000001

E isooo

10000

6000

0

Stimulators (LOL)

Fig. 6 Proliferative response of the C3/5 clone against the BCR1/25

peptide. In A, the C3/5 clone (5 X iO� cells/well) was cultured for 24,

48, and 72 h with irradiated (8000 rad) autologous DE.LCL (l0�

cells/well) in the absence (�) or presence of 2 p.M (In) and 20 p.M (U)of peptide BCR1/25 (UI. lymphocytes cultured in medium alone). In B,

the C3/5 clone (5 X l0� cells/well) was cultured for 24 h with irradiated(8000 rad) autologous DE.LCL or ailogeneic SR.LCL, FR.LCL, and

MM.LCL (l0� cells/well) in the presence of 5 p.M (�), 20 p.M (5), and

50 p.M (U) or in the absence (Eli) of BCR1/25 peptide. LCLs at 5 X 106

cells/ml were incubated with the peptide for 30 mm before irradiation,

cultured for an additional 1 8 h with [3H]dThd, and harvested. cpm

values represent the means of three replicates. Bars, SD.

effect, peptide-pulsed FR.LCL cells were admixed with labeled

FR.APL cells at a I : I ratio before incubation with the C3/5

effector clone. Even under these conditions, C3/5 cells were

unable to lyse FR.APL cells (Fig. 7). FR.APL cells proved

otherwise sensitive to lysis as evidenced by the ability of LAK

cells to nonspecifically lyse these cells (Fig. 7). Identical results

were obtained using leukemic cells from patient P. G. and

increasing the incubation time to 6 h (data not shown).

DE SR FR MM

598 Immune Recognition of the pmIIRAR-a Protein

Table 1 Antigen pr esentation capability of A PL patients’ PBL�

Type of APCs -L- BCR 1/25 - BCR 1/25

DE.LCL J�(05 ± 162 3,161 ± 320

DE.PBL 18,083 ± 2,607 6,067 ± 360SR.PBL 16,556 ± 3,995 5,638 ± 941

FR.PBL 18,650 ± I 13 4,645 ± 847

MM.PBL 15,178 ± 3,659 4,210 ± 266PG.PBL 18,025 ± 1,856 3,541 ± 188

“ C3/5 cells (50,000/well) were cocultured with 10� irradiated

APCs incubated with BCR 1/25 (25 p.M) or PBS. Plates were culturedfor 48 h, labeled with [3H]dThd for additional 18 h, and harvested.Values represent the mean X SD cpm obtained in three replicates. C/S

cells incubated in medium alone incorporated 195 ± 70 cpm; irradiated

APCs incorporated 419-841 cpm.

. 1r�;:�:=r-�w:�_ -V

3:1 6:1 12�1 24:1.

EP?ector Target ratio

Fig. 7 Cytotoxic activity of clone C3/5 against allogeneic FR.APL

blasts before and after incubation with IFN-�y (10� units/ml, 48 h) in the

presence (#{149},#{149},Y, and A) or absence ( 0 , 0, E, V. and A) of the

BCRI/25 peptide. The target cells used were: FR.LCL (0 and #{149}),FR.APL (V and Y), FR.APL + FR.LCL (E and U), FR.APL + IFN-’y(A and A), and LAK + FR.APL ( <)).

PHA and TI Responses of Patients’ PBLs. Because of

the negative results obtained with lymphocytes from APL pa-

tients, a PHA response assay was performed to test the T-cell

response in these patients. Results are presented in Table 2 and

show that, compared to PBLs from two normal donors (D. E.

and C. H. R.), patients’ PBLs showed a dramatically reduced

response. These results were surprising to us, since all patients

were studied while in remission and off treatment for >6 months

(F. R. was in complete remission and off therapy for >2 years).

Comparable results were obtained when response to a specific

antigen (IT) was evaluated. Six-day proliferative responses to

IT averaged 293,959 ± 42,793 (SD) cpm in three donors

(including D. E. and C. H. R.) and 49,602 ± 15,047 cpm in the

patient group (P = 0.002).




4 C. Gambacorti-Passerini, unpublished results.

Table 2 PHA response in four APL patients and two healthy donors

Type of cells With PHA Without PHA

DE.PBL 20,528 ± 2613 107 ± 87

SR.PBL 3,281 ± 48 228 ± 97

FR.PBL 8,381 ± 476 171 ± 56MM.PBL 1,930 ± 100 214 ± 59

PG.PBL 9,150 ± 1,987 326 ± 83

C1-LR.PBL 26,547 ± 2,438 335 ± 101a Cells were thawed, washed, and seeded at l0� cells/well (see

‘ ‘Patients and Methods’ ‘ ). Values represent mean cpm ± SD from sixreplicates.

Screening of the pmIIRAR-a Fusion Region for HLA

Class I-binding Motifs. Although donor lymphocytes can

recognize pml/RAR-a peptides in a DR-restricted fashion, at-

tempts at generating class I-restricted CTLs in two donors (and

in two patients) failed. We recently obtained the binding motifs

for the 35 most frequent class I alleles and matched these motifs

against the sequence of the pmiIRAR-a fusion region. No good

matching was identified,4 suggesting the lack of binding motifs

for the class I HLA molecules most frequently encountered in

the general population. To provide partial confirmation to these

negative findings, BCR1/9 peptides were separately used in

HLA stabilization experiments. Different cell lines (see “Pa-

tients and Methods’ ‘) were used, enabling us to assess binding

to HLA-A2,3, B5101,63, and Cw0701,0702. Appropriate posi-

tive control peptides (natural ligands) were included and in-

duced an increase in the mean channel of fluorescence of 150%

(A0201 ), 500% (A0301 ), and 269% (Cw0702). All BCR 1/9

peptides induced increments of <20% (on no increase at all)

oven values obtained with control unrelated peptides.

DISCUSSION

The data presented here demonstrate specific proliferation,

cytotoxicity, and cytokine production by HLA-DR1 1 � donor

CD4 1-cell clones upon challenge with HLA-DR1 I LCLs or

PBLs pulsed with the BCR1/25 peptide; no BCR1/9 peptide-

specific CD8IHLA class I-restricted response could be gener-

ated in both donors and patients. We also showed that these

clones could not be obtained in four HLA-DR1 I � APL patients,

and that APL blasts from DRI 1 patients were not recognized by

a DR1 1-restricted CD4 1-cell clone and were unable to present

peptide BCR1/25. These results raise several questions regard-

ing the possible recognition of the APL-specific pml/RAR-a

molecule by I cells. On one side, our results confirm previous

findings that donor I cells can recognize a peptide encompass-

ing the fusion region of the pmIIRAR-a molecule (6). Previous

data indicate that the cellular processing of the pml/RAR-a

protein can produce peptides that stimulate anti-BCR1/25 clones

(6). Here, we show that BCR1/25-specific 1-cell clones can also

exert a specific cytotoxic activity and release cytokines upon

stimulation with the peptide. We also present evidence that

cytotoxic activity and TNF-a release by these clones are sepa-

rate functions, although both seem to be BCR1/25 specific.

Finally, LCLs and PBLs derived from APL patients expressing

the relevant restriction element (HLA-DR I 1 ) are able to present

BCR1/25 similar to autologous LCLs.

On the other side, two different sets of data have to be

considered. First, repeated attempts at generating anti-BCR1/25

1-cell clones in four DRI 1 � APL patients were not successful.

These results cannot be attributed to an insufficient antigen-

presenting capability in these patients, since their LCLs and

PBLs induced proliferation and cytotoxicity in the C3/5 clone in

the presence of BCR1/25 similar to autologous LCLs. However,

it is possible that the frequency and/or the responsiveness of

anti-BCRI/25 lymphocytes was particularly low in these pa-

tients, even if PBLs were recovered when the patients were in

remission with normal peripheral blood counts and 1-cell sub-

sets values. The results of the PHA and IT response assays

strongly support this conclusion and indicate that a generalized

impairment of the cellular immune system, already reported in

cancer patients (15), can be present even during long-term

remissions. The molecular analysis of the observed hyporespon-

siveness is presently under investigation in our laboratory.

The possible deletion or suppression of anti-pmiIRAR-a-

specific T-cell clones by APL cells is another possibility that

deserves further investigation as well as ways to increase the in

vivo frequency of anti-pmlIRAR-a precursors in APL patients.

The failure to generate anti-BCRI/25-specific clones in APL

patients represents the most relevant problem to be solved

before therapeutic approaches can be considered.

Second, in both APL patients and healthy donors we were

unable to raise CD8, HLA class-I restricted 1-cell clones. This

is of particular importance given the usual and strong suppres-

sion of the DR gene expression in APL cells. Although our

strategy of pulsing APCs with short peptides could not have

been successful for technical reasons, it produced positive ne-

sults in other systems (16) and against known immunogenic

peptides. It has been demonstrated that inhibitory peptides com-

petitively block the binding to HLA molecules of stimulatory

peptides (1 7). Since a mixture of 9-men peptides was used in our

experiments, peptide cross-inhibition cannot be excluded. The

use of different stimulation protocols (e.g., using pml/RAR-a-

transfected cells) or APCs (like dendritic cells) could produce

positive results. These results can also be explained by the

observed lack of effective binding motifs for HLA class I

molecules in the pm1IRAR-a junction. A recent article by

Bocchia et al. (18), analyzing HLA binding to two pm1IRAR-a

peptides, is consistent with such an hypothesis.

Thus, it is possible that in APL, the pmlIRAR-cz protein is

recognized by T lymphocytes, but only when presented by a

restriction element (HLA-DR1 1) that is not present on the APL

cells themselves. The recognition of putative DR� APL stem

cells in the form of colony-forming units could still be possible.

However, the existence of these cells has yet to be demonstrated

(19), and true APL colonies are difficult to obtain (20). Alter-

natively, the induction of DR molecules on APL cells could

permit an efficient immune recognition (20).

The lack of expression of DR molecules by APL cells and

the resulting inability of DRI 1 � APL cells to present pml/

RAR-a peptides could also be reflected by the lack of clinical

significance of DR I 1 expression in APL patients undergoing

bone marrow transplantation. In collaboration with the Intema-



600 Immune Recognition of the pml/RAR-a Protein

6. Gambacorti-Passerini, C., Grignani, F., Arienti, F, Pandolfi, P. P.,Pelicci, P. G., and Parmiani, G. Human CD4 lymphocytes specifically

tional Bone Marrow Transplant Registry, an analysis was con-

ducted in I 89 APL patients in first remission who underwent an

ailogeneic related bone marrow transplant. The patients were

grouped into HLA-DR1 1 � (n 44) and HLA-DR1 1 (n =

145). No significant difference in survival or disease-free sun-

vival was evident between the two groups. Also, the proportion

of DRI 1 patients (23%) does not differ significantly from that

seen in the general population.4 In case the pmlIRAR-a junction

could not be presented by the HLA class I molecule, then the

only chance that this fusion protein can be recognized in APL

cells by I lymphocytes resides in the restoration of DR expres-

sion through pharmacological (2 1 ) or genetic manipulation, or

in the targeting of the still hypothetical DR� APL stem cells.

Our studies demonstrate that the BCR1/25 peptide corre-

sponding to the fusion region of the pm1IRAR-a protein present

in APL cells can be specifically recognized by donor (but not by

patient) CD4 1 cells in a HLA class II (DR1 1)-restricted fash-

ion; CD8IHLA class I-restricted I cells were not generated,

possibly due to the lack of HLA-binding motifs in the pml/

RAR-cx fusion region. Finally. APL blasts were not lysed by

anti-pml/RAR-a cytotoxic T cell clones due to the lack of

expression of the DR1 I molecule by the APL blasts studied.

Further immunotherapeutic approaches to APL will need to

overcome the two main problems encountered in this study: the

lack of DR expression in APL cells (20) and the poor immune

status of APL patients.

ACKNOWLEDGMENTS

We thank Dr. H. G. Rammensee for providing the HLA-binding

motifs and for critical reading of the manuscript, and Dr. M. M.Horowitz, Dr. P. A. Rowlings, and Dr. M. M. Bortin (International BoneMarrow Transplant Registry, Milwaukee, WI) for providing survival

data analysis on transplanted APL patients. We also thank the Antony

Nolan Foundation (London, England) which kindly provided the CHRcells, Dr. M. T. Illeni for HLA class I typing, Dr. F. Poli (Ospedale

Maggiore, Milan, Italy) for DR typing, and T. Blesken for technicalassistance in the cytokine detection assays.

REFERENCES

1. Rowley, J. D., Golomb, H. M., and Dougherty, C. 15/17 transloca-tion, a consistent chromosomal change in acute promyeiocytic leukemia.

Lancet, 1: 549-550, 1977.

2. dethe, H.. Chomienne, C., Lanotte, M.. Degos, L., and Dejean, A.The t(15;l7) translocation of acute promyelocytic ieukaemia fuses theretinoic acid receptor a gene to a novel transcribed locus. Nature

(Lond.), 347: 558-561, 1990.

3. Borrow. J., Goddard, A. D., Sheer, D., and Solomon, E. Molecular

analysis of acute promyeiocytic leukemia breakpoint cluster region onchromosome 17. Science (Washington DC), 249: 1577-1580, 1990.

4. Grignani. F., Fagioli, M., Alcalay, M., Longo, L., Pandolfi. P. P.,

Donti, E., Biondi, A., Lo Coco, F., Grignani, F., and Pelicci, P. G. Acutepromyelocytic leukemia: from genetics to treatment. Blood, 83: 10-25,

1994.

5. Gambacorti-Passenini, C. Immunogenicity of fusion proteins: an cx-ample of tumor-specific/transformation-related antigens. Int. J. Clin.Lab. Res., 23: 186-191, 1993.

recognize a peptide representing the fusion region of the hybrid proteinpml/RARa present in acute promyelocytic ieukaemia cells. Blood, 81:

1369-1375, 1993.

7. Chen, W., Peace, D. J., Rovira, D. K., You, S-G., and Cheever, M. A.

T-cell immunity to the joining region of p210BCRABL protein. Proc.Nati. Acad. Sci. USA, 89: 1468-1472, 1992.

8. Gillis, S., Ferm, M. M., Ou, W., and Smith, K. A. 1-cell growth

factor: parameters of production and quantitative microassay for activ-ity. J. Immunol., 120: 2027-2032, 1978.

9. Carballido, J. M., Carballido-Perrig, N., Terres, 0., Heusser, C. H.,and Blaser, K. Bee venom phospholipase A2 specific T cell clones fromhuman allergic and non allergic individuals: cytokine patterns change inresponse to the antigen concentration. Eur. J. Immunol., 22: 1357-1363,1992.

10. Andersson, U., Andersson, J., Lindfors, A., Wagner, K., Moller, G.,and Heusser, C. H. Simultaneous production of interleukin 2, interleukin4 and intenferon--y by activated human blood lymphocytes. Eur. J.

Immunol., 20: 1591-1596, 1990.

11. Gambacorti-Passerini, C., Rivoltini, L., Supino, R.. Mariani, M.,and Parmiani, 0. Differential lysis of melanoma clones by autologousrecombinant interleukin 2-activated lymphocytes. Relationship withspontaneous resistance to doxorubicin. mt. J. Cancer, 42: 544-548,

1988.

12. Salter, R. D., and Cresswell, P. Impaired assembly and transport of

HLA-A and B antigens mutants TxB cell hybrid. EMBO J., 5, 943-949,1986.

13. Steinle, A., and Schendel, D. J. HLA class I alleles of LCL 721 and174xCEM. T2 (T2). Tissue Antigens, 43: 268-270, 1994.

14. Dc la Salle, H., Hanau, D., Fricker, D., Urlacher, A., Kelly, A.,Salamero, J., Powis, S. H., Donato, L., Bausinger, H., Laforet, M., Jeras,M., Spehner, D., Bieber, T., Faikenrodt, A., Cazenave, J. P., Trowsdale,T., and Tongio, M. M. Homozygous human TAP peptide transportermutation in HLA class I deficiency. Science (Washington DC), 265:

237-241, 1994.

15. Mizoguchi, H., O’Sheea, J., Longo, D., Loeffler, C., McVicar, D.,and Ochoa, A. Alterations in signal transduction molecules in 1 lym-phocytes from tumor-bearing mice. Science (Washington DC), 258:

1795-1798, 1992.

16. Peace, D. J., Smith, J. W., Chen, W., You, 5-0., Cosand, W. L.,Blake, J., and Cheever, M. A. Lysis of ras oncogene-transformed cells

by specific cytotoxic I lymphocytes elicited by primary in vitro immu-nization with mutated Ras peptide. J. Exp. Med., 179: 473-479, 1994.

17. Gedde-Dahl, 1., III, Fossum, B., Eriksen, J. A., Thorsby, E., and

Gaudemack, G. T cell clones specific for p21 ras-derived peptides:characterization of their fine specificity and HLA restriction. Eur. J.Immunol., 23: 754-760, 1993.

18. Bocchia, M., Wenworth, P. A., Southwood, S., Sidney, J., McGraw,K., Scheinberg, D., and Sette, A. Specific binding ofleukemia oncogenefusion protein peptides to HLA class I molecules. Blood, 85: 2680-2685, 1995.

19. Turhan, A. G., Lemoine, F. M., Debert, C., Bonnet, M. L., Baillou,C., Picard, F., Macintyre, E. A., and Varet, B. Highly purified primitivehematopoietic stem cells are PML-RARA negative and generate non-

clonal progenitors in acute promyelocytic leukemia. Blood, 85: 2 154-

2161, 1995.

20. Jinnai, I. In vitro growth response to G-CSF and GM-CSF by bonemarrow cells of patients with acute myeloid leukemia. Leuk. Res., 14:

227-240, 1990.

21. Khanna-Gupta, A., Kolibata, K., Zibeilo, T., and Berliner, N. NB4

cells show bilineage potential and an aberrant pattern of neutrophilsecondary granule protein gene expression. Blood, 84: 294-302, 1994.



1996;2:593-600. Clin Cancer Res S Dermime, C Bertazzoli, E Marchesi, et al. promyelocytic leukemia patients.pml/RAR-alpha hybrid protein by lymphocytes of acute Lack of T-cell-mediated recognition of the fusion region of the

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Lack of T-Cell-mediated Recognition of the Fusion Region of the … · Vol. 2. 593-600. Marc/i 1996 Clinical Cancer Research 593 Lack of T-Cell-mediated Recognition of the Fusion