0022-1 767/93/1505-2046$02.00/0
The Journal of Immunology
Copyright 0 1993 by The American Association of Immunologists
VOl 150, 2046-2055, No. 5, March 1, 1993
Printed in U.S.A.
Human Peripheral y6 T Cells Recognize hsp60
Molecules on Daudi Burkitt's Lymphoma Cells'
lndreshpal Kaur,* Stephan D. Voss,* Radhey S. Gupta,' Kathleen Schell,* Paul
Paul M. SondeI2*§¶
Fisch,* and
Departments of *Human Oncology, §Pediatrics and ¶Genetics, University of Wisconsin, Madison, WI 53792, tDepartment
of Biochemistry, McMaster University, Hamilton, Ontario L8N 325, Canada, and +Laboratory of Molecular Biology, Hills
Road, Cambridge, CB2 2QH, England
ABSTRACT.
Studies with the use of polyclonal rabbit antiserum reactive with 60-kDa heat shock proteins (hsp60)
suggested that hsp60-related molecules could be found on the surface of Daudi cells and were involved in their
recognition by certain human yS T cells. The present study confirms this finding by using a mAb specifically
recognizing hsp60. This mAb can block outgrowth ofhuman y6 T cells in response to stimulation with Daudi and
in response to an extract of the mycobacteria H37Ra. This anti-hsp60 mAb stains the surface of Daudi cells, but
does not stain either Raji or EBV-transformed B cells, cells which do notstimulate y6 T cell outgrowth. Anti-hsp60
mAb could immunoprecipitate
a 60-kDa molecule from
the H37Ra extract but was unable toprecipitate this 60-kDa
molecule if the rnAb was first absorbed on Daudi cells. This rnAb also precipitated a 60-kDa molecule from the
surface of Daudi cells which shows an electrophoretic mobility pattern consistent with hsp60. These experiments
demonstrate that human y6 T cells recognize hsp60-related epitopes on the surface of Daudi cells and within
mycobacterial extracts. Journal of Immunology, 1993, 150: 2046.
S
ince the discovery of the minor subset of lymphocytes bearing the y6 TCR, investigators have been
searching for ligands recognized by y6 T lymphocytes. In the mouse, T cells expressing the y6 TCR are
found in theperipheral blood andare distributed at different
sites in the body such as the intestines (1, 2), epidermis
(3,4), and reproductive organs (5). Human y6 T cell clones
have been shown to exert nonspecific cytotoxicity against
a variety of tumor targets (6-9). This suggests that the y6
T cells may act as cytolytic effector cells to eliminate virally
Received for publication July 14, 1992. Accepted for publication December
10, 1992.
The costs of publication of this article were defrayedin 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 This research was supported by National Institutes of Health Grants and
Contracts CA-53441,
CA-05436,
CA-32685,
CM-87290,
CA-14520,
CA-13539, CM-47669, HL-02143, and
RR-03186; American Cancer Society
Grant CH-237; and National Cancer Institute Training Grant 5T32-CA09471
and a Lutheran Brotherhood/Life and Health Insurance Medical Research Fund
M.D./Ph.D. training fellowship to S. D. V.
Address correspondence and reprint requests to Dr. Paul M . Sondel, University of Wisconsin, Comprehensive Cancer Center, 600 Highland Ave., Madison. WI 53792-0001.
infected or tumor cells. Some human y6 T cell clones can
recognize defined Ag, suchas self Ig present on tumor cells
(10) or tetanus toxoid (1 1) in a MHC-restricted manner,
whereas several studies have shown human y6 T cells can
recognize tumor targets in a non-MHC-restricted manner
(7-9, 12, 13). Mycobacterial extracts have also been shown
to stimulate both human and murine y6 T cells (14-20).
Molecules related to the 65-kDa heat shock protein found
in mycobacterial extracts appear to be ligands recognized
by some murine as well as human y6 T cells (14, 19, 2 1,
22).
We have previously shown that human peripheral yS T
cells coexpressing the Vy9N62 TCR V region chains lyse
Daudi Burkitt's lymphoma cells in a non-MHC-restricted
manner but mediate minimal lysis of Raji Burkitt's lymphoma cells (12, 23, 24). These same Vy9N62 y6 T cells
also proliferate in response to Daudi cells but not to Raji
cells, suggesting that Daudicells, but not Rajicells, express
a ligand on their cell surface stimulatory for Vy9AJ62y6
T cells. These same y6 T cells proliferate in response to
mycobacterial extracts, in the presence of APC. Our earlier
experiments (23) showed that the proliferative response of
2046
Journal of Immunology
the y6T cells to Daudi cells and to the mycobacterial extract
could be inhibited by a polyclonal rabbit antiserum to the
mammalian groEL-related heat shock proteins (hsp60 or
hsp58). In the present study we confirm and extend these
findings by using a mAb specific for the hsp60 molecule
to characterize the molecules present on Daudi cells and in
mycobacterial extracts which are able to stimulate human
y6 T cells.
Materials and Methods
Cell lines
Daudi and Raji Burkitt’s lymphoma lines (obtained from
American Type Culture Collection (ATCC), Rockville,
Maryland) were maintained in supplemented RPMI 1640
medium containing 2 mML-glutamine, 100 IU/ml penicillin, 100 pg/ml streptomycin (all from Whittaker, Walkersville, MD), 25 mMHEPES buffer (Sigma Chemical Co.,
St. Louis, MO), and 10% FCS (HyClone, Logan, UT)
whereas the EBV-transformed B cell line, LCL 721, was
maintained in supplemented RPMI 1640 medium containing 10% human serum (Pelfreez, Roger, AK).
Antibodies
11-13 is a mAb of IgGl isotype, raised by immunizing mice
with recombinant human mitochondrial Hsp60 (Pl) (25,
26). The amino acid sequence of the P1 protein shows a
high degree of homology to that of the GroEL protein of
Escherichia coli, the 65-kDa major Agof mycobacteria
(Mycobacterium leprae, Mycobacterium tuberculosis, and
Mycobacterium bovis BCG) and to the ribulose 1,sbiphosphate carboxylase-oxygenase large subunit-binding
protein of plant chloroplasts. In one- and two-dimensional
immunoblots, the mAb 11-13 reacts only with the P1protein
from CHO and human cells. In all experiments 11- 13 used
was a culture supernatant rather than in the form of an
ascites fluid. As a negative control, the fusion partner used
to prepare the hybridoma secreting 11- 13, was cultured under the same conditions as the 11-13-secreting hybridoma,
and its supernatant collected and tested (designated “antibody control,” NSl). The mAb TiyA reacts with the Vy9encoded TCR y protein (kindly provided byT. Hercend,
Villejuif Cedex, France (27)), mAb BB3 reacts with the
V62-encoded TCR 6 chain (kindly provided by L. Moretta
and E. Ciccone, Genova, Italy (28)). FITC-labeled mAb
anti-human Leu-4 (reacts with the CD3 molecule on all T
cells), anti-human aP TCRl (reacts with T cells expressing
the aP TCR), anti-human y6 TCRl (reacts with T cells
expressing the yS TCR), goat anti-mouse, mouse IgG 1 and
PE labeled mAb anti-Leu-19 (reacts with the CD56 molecule on NK cells), and mouse IgGl (all fromBecton Dickinson, Mountain View, CA) were used for flow cytometric
analysis. Polyvalent anti-GroEL rabbit serum was kindly
provided byW. Welch, San Francisco (23). Anti-human
2047
class I1 DwDQ (CC 11.23) mAb was kindly provided
by R.
DeMars (29). The hybridoma (OKTll), producing mAb
against T1 I (CD2), was obtained from ATCC; mAb OKTll
was used as a culture supernatant.
Flow cytometry
Cells were stained with unconjugated mAb by using the
standard indirect immunofluorescence procedure and concentrations found to be discriminatory, as previously published (12). Goat anti-mouse-FITC wasusedasthe
secondary antibody. FITC- or phycoerythrin-conjugated mAb
were used in a single or double immunofluorescent staining, The stained cells were analyzed on a FACScan flow
cytometer (Becton Dickinson) by using Lysis I1 software.
Data were acquired in listmode, and analysis was limited
to viable cells which excluded propidium iodide (1 pg/ml)
as done previously (12, 23).
Flow cytometric proliferative outgrowth assays
PBL from healthy individuals were separated on Ficoll/
Hypaque. PBL ( lo6) were cultured in supplemented RPMI
1640 with10%FCS witheither 3 X lo5 irradiated (120 Gy)
Daudi or Raji cells or optimal concentrations of the mycobacterial extract (sonicated extract of the mycobacteria
H37Ra, kindly provided by Dr. K. Takayama,Madison WI)
in a 48-well tissue culture plate. Appropriate mAb were
added with the Daudi cells. Seven days later cells from all
the cultures were stained with the respective mAb and analyzed on the flow cytometer.
Western blot analysis
H37Ra extract was separated by electrophoresis, under reducing conditions on a 10% SDS-PAGE gel(30). The proteins were transferred to nitrocellulose under an electric
field of 20 mA overnight. The nonspecific staining was
blocked by treating the nitrocellulose with 5% BSA in PBS.
The nitrocellulose was stained with either 11-13 or NS1
culture supernatants. Rabbit anti-mouse coupled to horseradish peroxidase was used as a secondary reagent. The
enzyme color was developed with diaminobenzidine (0.5
mg/ml PBScontaining 8 p1 H202/mlPBS), and the reaction
was stopped by removing the substrate solution and washing the nitrocellulose with PBS.
Immunoprecipitation
From H37Ru extract. mAb 11-13 or NS 1 culture supernatant were coupled to Prot G-agarose beads (30). H37Ra
extract was addedto the mAbcoupled beads and rockedfor
2 to 3 h at 4°C. The unbound extract was washed out and
the protein bound to the antibody was eluted with 100 mM
glycine buffer, pH 2.5. The pH of the eluted protein was
neutralized with 1 Mphosphate buffer, pH 8.0. The eluted
H U M A N y8 T CELLS RECOGNIZE hsp60 MOLECULES ON DAUDI CELLS
2048
samples were run on a 10% SDS-PAGE gel and proteins
the
stained by silver staining (30). In some experiments, the
11-13 mAb was first absorbed on Daudi cells or Raji cells
or, alternatively, Daudi or Raji cells that had been treated
for 2 h at 4°C with aggregated rabbit serum (30 rnin at
65"C), in order to block the FcR on Daudi and Raji cells.
The absorptions were performedby incubating 0.5 ml of the
culture supernatant with 2 X lo7 cells at 4°C for 30 min.
The cells were spun and the culture supernatant removed
to repeat the absorption on fresh cells three times more to
achieve complete absorption of the mAb on cells.
From surface radioiodinated Daudi cells. Radioiodination of Daudi cells was performed as described previously (23, 31). Daudi cells were radioiodinated with Iodogen (Pierce Chemicals, Rockford, IL). Daudi cells (5 X
lo7) were resuspended at 2 X lo7 cells/ml in HBSS. Samples of 5 mCi Na'251 (Amersham, Arlington Heights, IL)
were added to cells without camer NaI. Aliquots of 500 pl
of cells
1251 were added to glass tubes coated with 150
pg Iodogen. After 30 min at room temperature cells were
washed three times with HBSS and lysed in NP-40 lysis
buffer (0.5% NP-40, 140 mM NaCl, 5 mMEDTA, 100
pg/ml PMSF, 10 pg each leupeptin, aprotinin, and antipain
(Sigma)). After 30 min on ice, lysates were cleared by centrifugation for 20 min at14,000 rpm ina microfuge. Lysates
were preincubated with both nonspecific mouse IgG agarose (Sigma) and protein G-Sepharose (GammaBind Plus
Protein G beads, GENEX, Gaithersburg, MD) followed by
incubation for 7 h with GammaBind Plus Protein G beads
(GENEX) that hadbeen precoated with either 2 ml of 11- 13
mAb or 2 ml ofantibody control NS 1,or 10 pl of polyclonal
rabbit serum against hsp58 (23), the mammalian homolog
of GroEL. Immunoprecipitates were washed extensively,
followed by addition of 7.5 pl of boiling buffer (20 mM
Tris, pH 7.5,5% 2-ME, 0.3% SDS) and heating at 95°C for
2 to 3 min. Precipitates were eluted from beads into 120 pl
IEF sample buffer (9.5 M urea, 2% w/v NP-40, 1.6% pH
5-7 Ampholines (Pharmacia), 0.6% pH3.5-10Ampholines, 5% 2-ME) and analyzed by two-dimensional IEF/
SDS-PAGE. Samples of 5 pg bovine muscle actin (Sigma)
were added to each sample as an internal IEF standard
(PI = 5.4-5.5, m.w. = 45 m a ) .
Two-dimensional IEF/SDS-PAGEwas performed according to the method of O'Farrell(32), with modifications
by Jones (33). Samples were loaded on 15 cmIEF tube gels
(9.2 M urea, 4% acrylamide, 2% w/v NP40, 1.6% pH 5-7
ampholines, and 0.6% pH 3.5-10 ampholines) that had
been prefocused at constant 2 mA until 8OOV was reached.
IEF was for 14,000 V-h at 800 V, followed by 2000 V-h at
1000 V. Tubes wereextruded into SDS sample buffer (62.5
mM Tris, pH 6.8, 10% w/v glycerol, 2.3% SDS, 0.002%
bromphenol blue) and stored at -20°C until second dimension gels were run. For the second dimension, tubes were
thawed quickly at 37"C, layered onto 10% polyacrylamide
+
gels, and electrophoresed at 30 mA constant current per gel.
After electrophoresis, gels were stained with Coommassie
blue, fixed, dried, and exposed to autoradiographic film.
Results
Anti-hsp6O rnAb, 11-1 3 blocks proliferative responses
of y6 T cells to Daudi cells and to the mycobacterial
extract
Previously published reports from our laboratory (23) suggested that there are GroEL (stress proteins in E. coli) related molecules on Daudi cells which could beinvolved in
stimulating human peripheral y6 T cells. Those studies
were performed by using a polyclonal rabbit antisera specific for the mammalian GroEL-related heat shockproteins.
To clarify the precise nature of the molecules involved, in
the present study we have used a mAb, 11- 13 raised against
recombinant hsp60 (PI) (25, 26).
PBL from healthy donors were stimulated by either irradiated Daudi cells or the H37Ra extract, alone or in the
presence of 11- 13 mAb or the antibody control supernatant
NS1. Seven days later the cultures were analyzed for expansion of y6 T cells by flow cytometry. The 11-13 mAb
blocked the stimulation of y6 T cells whereas the NS1 antibody control did not (Fig 1). The percentage of y8 T cells
increased from 2.0% (in medium alone) to 21.6% when
stimulated by irradiated Daudi cells. Similarly, thepercentage of y6 T cells increased from 2.0% (in medium alone)
to 15.5% when stimulated by H37Ra extract. When PBL
were stimulated with Daudicells in the presence of the 11- 13
mAb the % of y6 T cells increased to only 4.1% (corresponding to 81% inhibition compared to the response to
Daudi cells in the absence of the 11-13 mAb). The response
to the mycobacterial extract in the presence of the mAb
11- 13 induced only 4.0% y6 T cells (corresponding to 74%
inhibition). Similar responses were observed when PBL
from two other donors were used as the responding cells
(data not shown).
11-1 3 rnAb stains the surface of Daudi cells
As the 11- 13 mAb blocked the proliferative response of
peripheral y6 T cells to Daudi, we investigated if this mAb
detected molecules on the surface of Daudi cells by immunofluorescence. Prior studies and the results shown in
Figure 1 demonstrate that Daudi cells, but not Raji cells or
EBV-LCL cause proliferation of the y6 T cell population
(23, 24). When both Daudi and Raji cells were examined
by flow cytometery for cell surface staining with 11-13
mAb, only Daudi cells were stained with this mAb. The
intensity of staining on Daudi cells with 11-13 mAb was
brighter than the staining seen with the isotype control antibody (mAb against OKTl 1). In contrast, the 11-13 mAb
did not stain the surface of Raji and 721 cells (Fig. 2). This
lournal
2049
of
B
stimulus only
stimulus+NSl
control
daudi
H37Ro
medium
stimulus
FIGURE 1. Proliferation of peripheral y8 T cellsstimulated by Daudi cells or themycobacterial extract (H37Ra) in the
absence and presence of monoclonalantibodies, 11-13, raised against hsp60 (PI) and the antibody control (NSI). PEL (1 0') from
ahealthy donor were cultured with
irradiated Daudi cells (3 x 105/ml) or appropriate dilutions of the H37Ra extract as
determined earlier. 11-13 mAb or the control mAb were added at a dilution of 1/2. Seven days later, the cells were stained by
direct immunofluorescence with anti-yS TCR mAb coupled to FlTC and analyzed on FACScan. For each specimen, 10,000
events were analyzed. Only viable cells, as determined by exclusion of propidium iodide (0.5 p g h l ) , were included in the
analysis. Nonspecific binding was controlled for by using appropriate isotype controls, to determine gating for positivity.
staining was reproducible in three other experiments performed separately. The staining on Daudi cells shows the
presence of molecules on the Daudi cell surface that are
immunologically cross-reactive with hsp60, as recognized
by the mAb 11- 13. Because this same 11-13 mAb also inhibited the proliferative responses of y6 T cells to Daudi,
this also suggests that the molecules recognized by 11-13
mAb on Daudi cells are involved in the stimulation of peripheral y6 T cells.
Specificity of y6 T cell inhibition by 11-13 mAb
The inhibitory effect of the 11-13 mAb on y6 T cell expansion was not due to a toxic effect of this antibody on
either the stimuli (i.e., Daudi cells), or on the responding
cells. The total number of cells recovered from these cultures, either with or without treatment with 11- 13 mAb, did
not change (Table I) and was greater than the recovery of
PBL cultured in the absence of Daudi cells (i.e., in medium
alone). In addition to the y6 TCR, other surface markers
(CD3, CD4, CD8, CD56, and T cells expressing the aP
TCR) were also studied in these cultures (Table I). The
addition of 11-13 mAb to the Daudi stimulated cultures
causes the percentage of y6 T cells to increase only to 2.8%,
vs 21.6% in the
absence of mAb 11-13, while there is a
compensatory increase in the percentage of CD8+ cells
(30.7% vs 22.0%). The outgrowth of the Vy9lV62 subset
(the common subset of the y6 T cells in the peripheral
blood) in response to Daudi cells was blocked by the 11- 13
mAb, whereas the antibody control supernatant, NS 1, had
no effect on the outgrowth of Vy9lV62 T cells in response
to Daudi cells (Table I). These data indicate that 11- 13 mAb
does not inhibit outgrowth of all responding cells in response to Daudi, but rather preferentially blocks the response of y6 T cells.
As the 11- 13 mAb binds to Daudi cells (Fig. l), we then
investigated whether other mAb that bind to Daudi cells
2050
HUMAN y8 T CELLS RECOGNIZE hsp60 MOLECULES ON DAUDI CELLS
of 58 kDa (Fig. 3, lane b ) and a less prominent, related
60-kDa molecule,indicating that 11-13 mAbdoes recognize
forms of an approximately 60-kDa moleculein the H37Ra
extract. Lane a in Figure 3 shows that the NS1 antibody
control does not appear to stain anything in the H37Ra
extract.
11-1 3 mAb recognizes a 60-kDa molecule in H37Ra
extract which cross reacts with a 60-kDa molecule
on the surface of Daudi cells
Based on the finding that 11-13 mAb stains the Daudi cells
as well as recognizes hsp60-related molecules within the
H37Ra extract, we tested whether these molecules were
related to each other. The 11- 13 mAb was coupledto Prot-G
agarose and the moleculein the H37Raextract recognized
and boundby 11-13 mAb waseluted and separated on a 10%
SDS-PAGE gel. Lane c in Figure 4 shows that 11- 13 mAb
could precipitate an approximately 58- to 60-kDa size molecule from the H37Ra extract.
The identity of this molecule
as hsp60 was confirmed by western blot analysis demon100
10’
lo2
lo3
104
strating reactivityof this band with the
11-13 mAb (data not
Log Green Fluorescence
shown). The separate band, labeled as H in Figure 4, lanes
FIGURE 2. Surface staining of Daudi, Raji, and 721 cells
c-g, stained with a rabbit antibody against mouse Ig, conwith 11-13 mAb 1-(
and an irrelevant isotype control mAb,
firming that this separate band is the H chain of the mAb
anti-OKT11 (- - - - -) l o 5 cells were stained with the respecused in the precipitations, whereas the 58-to 60-kDa band
tive rnAb by indirect immunofluorescence by using goat-antiabove it in the same lanes is the
specific hsp60 protein
mouse IgG coupled to FlTC as the secondary reagent and
eluted
with
the
11-13
mAb.
analyzed on a FACScan analyzer. For each sample, 10,000
To demonstratecross-reactivitybetween the molecule
on
events were analyzed. Only viable cells, as determined by
Daudi cells recognized by the
11- 13 mAb and the molecules
propidium iodide exclusion (0.5 pg/ml), were included inthe
analysis.
precipitable by11-13 mAb from the H37Ra extract, the
11-13 mAb was preabsorbed on Daudior Raji cells (which
had been either pretreated or not treated with aggregated
would also block stimulationof y6 T cells by Daudi. CC
11.
normal rabbit serumto block the FcR).The absorbed 11- 13
23, a mAb against MHC class I1 molecules stains Daudi
mAb preparation was then coupled to Prot-G agarose to
cells (Daudi cells have class I1 molecules on them but do
immunoprecipitatemoleculesfromtheH37Ra
extract.
not have complete class I molecules as these cells are deWhen the eluate was separated on a 10% SDS-PAGE gel
ficient in &-microglobulin chains (34-38)). Yet when this
and stained for proteins (Fig. 4), the 60-kDa molecule that
antibody was added to cultures of PBL with Daudi cells,
hadbeenboundby
the unabsorbed 11-13 mAbwasno
there was no
inhibition of the y6 T cell outgrowth, but there longer seen when 11-13 mAb was first absorbed on Daudi
was a slight inhibition of CD4+ cell outgrowth, likely due
cells (lane b). Furthermore, absorption of mAb 11-13 on
to inhibition of MHC class I1 alloantigen recognition by
Daudi cells pretreated with aggregated rabbit serum also
CD4+ cells. This suggests that 11-13 mAb inhibits recogeliminated the ability of the mAb to bind the 60-kDa molnition of Daudi specifically by y6 T cells by binding to a
ecule (lane a ) , indicating that the absorptionof mAb 11-13
specific molecule on Daudi cells.
was not simplydue to interaction of this mAb with the FcR.
In contrast, if the 11-13 mAb was absorbed on Raji cells
11-1 3 rnAb also recognizes a 60-kDa molecule in the
(which were similarly treated or not treated with aggregated
mycobacterial extract
normal rabbit serum), it could still bind the 58- to 60-kDa
molecule from the H37Ra extract. This suggests that the
To determine which molecule within the H37Ra extract was
11-13 mAbdid specifically bind to some molecules on
recognized by 11- 13 mAb, we performed western blotting.
The H37Ra extract was separated on a 10% SDS-PAGE gel Daudi cells which were absent on Raji cells. The binding
and transferredto nitrocellulose paper and Western blotted of mAb 11-13 to Daudi cells could not have been to FcR,
present on Daudi cells, as the mAb 11-13 was able to bind
with 11-13 mAb and the antibody control, NS 1. The 11-13
to Daudi cells which had been treated with
aggregated normAb specifically detects a protein of an approximate size
Journalof Immunology
2051
Table I
Analysis of various cell populations after the
PBL
were stimulated with Daudi cells"
PEL Stimulated by:
Cell Surface Marker"
Daudi
CD3'
yS TCR'
Vy9N62'
CD4'
CD8'
CD56'
ap TCR'
absolute lymphocyte counts recovered
Daudi
NSl
Medium
85.2
1.9
1.7
56.7
22.0
5.3
80.0
0.48 x 10'
82.4
21.6
10.8
44.5
15.2
7.7
61.8
2.3 x lo6
Daudi +
11-13
Daudi +
CCll.23
86.2
2.8
1.9
41.7
30.7
7.4
87.2
2.6 x 10'
79.6
25.7
ND
28.5
21.2
13.5
53.9
2.4 x 10"
+
83.0
16.0
9.8
48.4
17.0
12.5
63.6
2.6 x 10'
In some wells 11-1 3 mAb or antibody control (NS1) or CCll.23, an anti-class II (DIUDQ)mAb were added. PEL from healthy donors were cultured for 7 days
with irradiated Daudi cells as described in Figure 1. The recovered cells were counted and analyzed for percentageof cells which are yS+, CD4', CDW, CD56+,
and T cells expressing theup TCR. This wasdone by immunofluorescence using appropriate mAb coupled to either FlTC or phycoerythrin. A total of 10,000 events
were analyzed and only viable cells as determined by propidium iodide exclusion were included in the analysis. Nonspecific binding was controlled for by using
appropriate isotype control antibodies to determine gating for positivity. Absolute lymphocyte counts are given for the cells recovered.
'I For each cell surface marker, the values given are the percentage positive cells from that culture.
mal rabbit serum. This clearly shows that there are molecules on the surface of Daudicells which are recognized by
11-13 mAb and that these molecules are immunologically
cross reactive to the 60-kDa molecules in the H37Raextract
recognized by the same mAb.
Characterization of the molecules on the Daudi cell
surface recognized by 11-13 mAb
To better characterize the molecules on Daudi cells that
were reactive with 11-13 mAb, we surface labelled Daudi
cells with '251 (23, 31). These surface-labeled Daudi cells
were then solubilized and immunoprecipitated with 11-13
mAb. The precipitates were analyzed by two-dimensional
IEF/SDS-PAGE (Fig. 5 ) . A parallel immunoprecipitation
was performedby using a polyclonal antiserumto the mammalian GroEL related hsp (23). Both the11-13 mAb andthe
anti-hsp58 antiserum immunoprecipitatedmolecules witha
molecular weight and a pattern of PI consistent with their
identificationas GroELhsp60-related proteins (23). In Figure 5 , b and c, the apparent double band in the SDS-PAGE
dimension migrating in the 60-kDa range most likely represents anartifact of this particular experiment as it was not
seen in other experiments (23). The hsp60 molecules precipitated from the Daudi cell surface by using either 11-13
mAb or the rabbit antiserum against the mammalian homolog of GroEL migrated as a collection of molecules of
identical m.w. but differing isoelectric point. It is possible
that these differences in PI are due to varying degrees of
glycosylation of the cell surface heat shock protein molecules, although we would thenexpect to see an increase in
m.w. corresponding to a more acidic PI. Instead, we favor
the interpretation that these molecules represent different
isoforms of a related family of hsp60 molecules. A similar
array of hsp60 molecules (as well as hsp70 molecules) with
multiple isoforms has been observed with heat shock proteins prepared from mitochondria (39). further supporting
a
b
kD
11697.4
-
66
1
45
-
FIGURE 3. Western blot analysisofthe H37Ra extractby
using 11-13 mAb (lane b) and the antibody control NS1 (lane
a). The position of the standard m.w. markers is indicated.
The mAb 11-1 3 identifies a molecule of approximate 58-kDa
size and weak detection of a closely related molecule.
the interpretation of multiple isoforms of a related family
of proteins rather than variableglycosylation. The basis of
these different isoforms is presently unclear (39).
When the polyclonal antiserum was used,
a 70-kDa molecule coprecipitated with the hsp60-related proteins (Fig.
5c). Although the experiments presented here do not directly test this, aninteresting hypothesis is that the70-kDa
molecule is a member of the hsp70 family of heat shock
hsp60 MOLECULES O N DAUDI CELLS
HUMAN y8 TRECOGNIZE
CELLS
2052
kD
-66
-H
-45
OW
H*
kD
116-
97.4
-
66 45 -
""
-L
-29
a
~
a ce
T
Y
FIGURE 4. Immunoprecipitation of molecules from H37Ra
extract by using 11-13 mAb (lanec), 11-13 mAb absorbed on
Daudi cells (lane b), on Raji cells (lane d), on Daudi cells
treated with aggregated rabbit serum (lane a), on Raji cells
treated with aggregated rabbit serum (lane e). Molecules immunoprecipitated by using NSl, the antibody control NSl
are shown in lane g and by using OKT11, an irrelevant isotype control mAb are shown in lane f. The position of the
standardm.w.markers
is indicated. H and L indicate the
position of the H and L chains of the mAb.
proteins that is in association with hsp60 molecules expressed at the cell surface. The association of hsp60 and
hsp70 molecules has been demonstrated with intracellular
heat shock proteins (40). This 70-kDa species was not detected with the hsp60-specific 11-13 mAb (Fig. 56), demonstrating that the molecule on the surface of Daudi cells,
involved in y6 T cell stimulation ismostlikely hsp60
related. These findings do not, however, rule out the possibility that an interaction between
hsp60 and 70-kDa molecules that could be disrupted by the 11-13 mAb, but unaffected by the polyclonal anti-hsp58 antiserum.
Discussion
The present study by using a mAb against hsp60 shows that
a 60-kDa molecule on the surface of Daudi cells and a
60-kDa molecule in the H37Ra extract are both recognized
by11-13, a mAb specific for hsp60.Furthermore, these
same 60-kDa molecules are each involved in the stimulation of human peripheral yi3 T cells. Previous studies have
indicated that 65-kDa heat shockproteins in the mycobacterial extracts can be ligands for human and murine y6 T
cells (14, 17, 18, 21, 22,41). The proliferative response of
the y6 T cells to mycobacterial extracts has been shownto
be primarily restricted to the Vy9N62 subset (23). The
proliferative triggering (23), blocking by anti-TCR or
anti"y6 antibodies (12), and the specific Vy9N62 usage
(23) noted when y6 T cells were cultured with the mycobacterial extract, all suggest that they6 TCR isrecognizing
the stimulatory determinant in the mycobacterial extract,
likely an hsp related molecule.
11697.4 -
~
-
I
66 -
45 -
11697.4 -
66 -
45 -
FIGURE 5. Two dimension gelanalysis of immunoprecipitation from surface radioiodinated Daudi cells by using the
antibody control NS1 (a), mAb 11-13 (b),and the polyclonal
rabbit antiserum raised against the mammalian homolog of
the GroEL protein of E. coli (c).As noted in the text, the array
of similarly sized proteins of varying pl is most likely due to
the presence of multiple isoforms of a related family of heat
shock proteins.
Our previous data, by using a polyclonal rabbit antiserum
against hsp60, suggested the presence of GroEL-related
molecules on the surface of Daudi cells and the possible
involvement of these in the non-MHC restricted stimulation
of peripheralyi3 T cells by Daudi cells. However, it remains
possible that different antibodies present in the polyclonal
antiserum may have been recognizing the active determinants on Daudi cells and in the mycobacterial extract.
The present study demonstrates that 60-kDa molecules
that are recognized by mAb11- 13 are present on thesurface
of Daudi cells and in the mycobacterial extract. The immunoprecipitations suggest that these 60-kDa molecules
are likelymycobacterialandmammalianheatshock
protein-related molecules. The mAb 11-13 bound to Daudi
cells (as determined in flow cytometric and absorption assays) whereas it did not bind to Raji cells. This binding
could notbe attributed to binding via theFcR as mAb 11- 13
absorbed equally wellto Daudi cells, that had been treated
with rabbit antiserum to block FcR.Immunoprecipitations
from the H37RA extract performed with mAb 11-13 absorbed on Daudi cells indicated that the molecules recog-
2053
journal of Immunology
nized by 11-13 on the Daudi cell surface and in the mycobacterial extract are immunologically cross reactive. These
P1- or hsp60-related molecules are not detectable by these
assays on the surface of Raji cells.
Pfeffer et al. (42) have shown that human y6 T cells are
triggered by low m.w. components (1-3 kDa) (possibly of
carbohydrate origin) of the mycobacterial extracts and that
the stimulation can be blocked by mAb to HLA
DR onclass
11-positive APC.They suggest that these structures are different from those present on Daudi cells capable of stimulating y6 T cells. However, we have previously shown
stimulation of y6 T cell clones in response to the mycobacterial extract by using APC devoid of all class I1 MHC
genes (23). Itmaybe that DR molecules are needed to
present the hsp related peptide, but not the intact 60-kDa
heat shock protein-related molecule. Endogenous heat
shock proteins of the hsp60 family expressed by lupus patients’ B cells have been shown to be involved in stimulating an autoreactive proliferation of the autoreactive y6
T cells isolated from patients with active lupus nephritis
(43). These T cell lines had the ability to augment the production of pathogenic anti-DNA autoantibodies that were
specific for the native DNA. The autoreactive response of
these y6 T cell lines was partially or completely blocked by
mAb to the 65-kDa heat shock protein, and not by mAb
against hsp70.
Heat shock proteins have been shown to be primarily
intracellular proteins (44). Nevertheless, the present studies
show that there are heat shock protein related molecules on
the surface of Daudi cells. Ferm et al. (45) have demonstrated induction of hsp60 expression in monocytic cell
lines by treating them with IFN-y or TNF-a. This expression of hsp60 was constitutive and could not be seenon the
cell surface. They have not been able see expression of
hsp60 on the surface of Daudi cells by using a mAb, ML-30,
raised against the mycobacterial hsp65 and cross-reactive
with mammalian hsp60. The present report with the use of
mAb 11-13 to identify hsp60-related molecules on the Daudi
surface by immunofluorescence as well as immunoprecipitation of surface iodinated cells, is in contrast to their findings but confirms and extends our prior studies (23).
Furthermore, in vitro cultured bone marrow-derived macrophages have shown surface staining with this same
ML-30 mAb 3 days after culture (46), and the surface expression of hsp60 on them was markedly increased after
IFN-y treatment for 9 days, indicating that the ML-30 mAb
(45) can recognize surface hsp60 related molecules, consistent with our present data.
Immunoprecipitations performed on cell lysates, prepared from surface-labeled Daudi cells, by using a polyclonal antibody against the mammalian homolog of GroEL
precipitated a 70-kDa protein along with a 58- to 60-kDa
protein, which might suggest that there is a 70-kDa surface
protein associated with the 60-kDa molecule. Alternatively,
identification of 70- and 60-kDa molecules could suggest
that the polyclonal antiserum contains antibodyagainst
other proteins that might be associated with the 60-kDa
molecule, as suggested by Ferm et al. (45). Within the cell,
the hsp60 molecules are known to be in association with the
hsp70 molecules which in turn act as chaperon molecules
to carry the hsp60 molecules from the nucleus to the mitochondria (39). We cannot be sure on the basis of the immunoprecipitations from Daudi cells by using polyclonal
anti-hsp60, that hsp60 related molecules on the surface of
Daudi cells are in association with hsp70 molecules. Unlike
the polyclonal anti-hsp60, the 11-13 mAb precipitates only
a 60-kDa molecule, and not a 70-kDa molecule from the
surface of Daudi cells, proving that a 60-kDa molecule
recognized by the 11-13 mAb is in fact on the cell surface.
It is possible that the 11-13 mAb somehow disrupted the
association of the 60- and 70-kDa molecules, thus preventing detection of a normally associated hsp70 molecule. Alternatively, there could besome antibodies in the polyclonal rabbit antiserum which recognize an epitope on the
distinct 70-kDa molecule which is not detected when we
used the 11-13 mAb. Further studies are needed to clarify
whether a 70-kDa molecule is actually associated with the
hsp related 60-kDa molecule recognized by 11- 13 mAb on
the Daudi surface. Nevertheless, these studies clearly demonstrate the presence of hsp60-related molecules on Daudi
cells immunologically cross reacting with the hsp60 molecules in the mycobacterial extracts, and that these hsp60related proteins, both in the mycobacterial extract and on
Daudi cells, are the molecules involved in the stimulation
of certain human peripheral y6 T cells.
Acknowledgments
We thank Drs. Hercend, Moretta. Ciccone, DeMars.
and Welch for proving
us with the antibodies and Dr. Takayarna for providing the mycobacteria.
References
1. Goodman,T., and L. Lefrancois. 1988. Expression of the
76-T cell receptor on intestinal CD8+ intraepithelial lymphocytes. Nature 333:855.
2. Bonneville, M., C. A. Janeway, Jr., K . Ito, W. Haser, 1. Ishida,
N. Nakanishi, and S. Tonegawa. 1988. Intestinal intraepithelial lymphocytes are a distinct set of y6 T cells. Nature 336:
479.
Koning, E , G. Stingl, W. M. Yokoyama, H. Yamada, W. L.
Maloy, E. Tschachler, E. M. Shevach, and J. E. Coligan. 1987.
Identification of a T3-associated yS cell receptor on Thy-]
epidermal cell line. Science 236:834.
Stingl, G., E Koning, H. Yamada, W. M. Yokoyama, E. Tshachler, J. A. Bluestone, G. Steiner, L. E. Same1con.A. M. Lew,
J. E. Coligan, and E. M. Shevach. 1987. Thy-1 dendritic
epidermal cells expressT3 antigen and the T-cell receptor y6
chain. Proc. Natl. Acad. Sci. USA 84:4586.
Lafaille, J. J., A. DeCloux, M. Bonneville. Y. Takagaki, and
S . Tonegawa. 1989. Junctional sequences of T cell receptor
+
+
2054
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
HUMAN y8 T CELLS RECOGNIZE hsp60 MOLECULES ON DAUDI CELLS
y8 genes: implications for y6 T cell lineages and for anovel
intermediate of V-(D)-J joining. Cell 59:859.
Bank, I., R. A. Depin Ho, M. B. Brenner, J. Cassimeris, E W.
Alt, and L. Chess. 1986. A functional T3 molecule associated
with a novel heterodimer on the surface of immature human
thymocytes. Nature 322:179.
Brenner, M. B., J. McLean, H. Scheft, J. Riberdy, A. SiewLan, J. G. Seidman, P. Devlin, and M. S. Krangel. 1987. Two
forms of the TCR y protein found on peripheral blood cytotoxic T lymphocytes. Nature 325-689.
Borst, J., R. J. Van de Griend, J. W. van Oostveen, A. SiewLan, C. J. Melief, J. G. Seidman, and R. L. H. Bolhuis. 1987.
A T-cell receptor ylCD3 complex found on cloned functional
lymphocytes. Nature 325:683.
Moingeon, P., S. Jitsukawa, E Faure, E Troalen, E Triebel,
M. Graziani, E Forestier, D. Bellet, C. Bolum, and J. Hercend.
1987. A y chain complex formsa functional receptor on
cloned human lymphocytes with natural killer-like activity.
Nature 325: 723.
Wright, A., J. E. Lee, M. P. Link, S. D. Smith, W. Caroll, R.
Lewy, C. Clayberger, and A. M. Krensky. 1989. Cytotoxic T
lymphocytes specific for self tumor immunoglobulins express T-cell receptor 6 chain. J. Exp. Med. 169.1557.
Kozbor, D., G. Trinchieri, D. S. Monas, M. Isobe, G. Russo,
J. A. Haney, C. Zmijewski, and C. Croce. 1989. Human TcR
y+S+ CD8+ lymphocytes recognize tetanus toxoid in an
MHC-restricted fashion. J. Exp. Med. 169:1847.
Fisch, P., M. Malkovsky, E. Braakman, E. Strum, R.L.H.
Bolhuis, A. Prieve, J. Sosman, V. A. Lam, and P. M. Sondel.
1990. ylS T cell clones and natural killer cell clones mediate
distinct patterns of non-major histocompatibility complexrestricted cytolysis. J. Exp. Med. 171:1567.
Van De Griend, R. J., W. J. M. Tax, B. A. van Krimpen, R.
J. Vreudgenhil, C. P. M. Ronteltap, and R. L. H. Bolhuis.
1987. Lysis of tumor cells by CD3+4-8-T-cell receptor apclones, regulated via CD3 and CD16 activation sites, recombinant interleukin-2, and interferon p. J. Immunol. 138:
162 7.
O’Brien, R. L., M. P. Happ, A. Dallas, E. Palmer, R. Kubo,
and W. Born. 1989. Stimulation of a major subset of lymphocytes expressing T cell receptor y8 by an antigen derived
from Mycobacterium tuberculosis. Cell 57t667.
Happ, M. P.,T. Kubo, E. Palmer, W. Born, and R. O’Brien.
1989. Limited receptor repertoire in a mycobacteria reactive
subset of yS T lymphocytes. Nature 342:696.
Born, W., L. Hall, A. Dallas, J. Boymel, T. Shinnick, D.
Young, P. Brennen, and R. O’Brien. 1990. Recognition of a
peptide antigen by heat shock-reactive gamma-delta T lymphocytes. Science 249:67.
Janis, E. M., S. H. E. Kaufmann, R. H. Schwartz, and D. M.
Pardoll. 1989. Activation of gamma delta T cells in the primary immune response to Mycobacterium tuberculosis. Science 244:713.
Augustin, A., R. T. Kubo, and G. K. Sim. 1989. Resident
pulmonary lymphocytes expressing the gamma/delta T cell
receptor. Nature 340:239.
Holoshitz, J., E Koning, J. E. Coligan, J. De Bruyn, and S.
Strober. 1989. Isolation of CD4-CD8mycobacteriareactive T lymphocyte clones from rheumatoid arthritis synovial fluid. Nature 339:226.
Kabelitz, D., A. Bender, S. Schondelmaier, B. Schoel, and S.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
H. E. Kaufmann. 1990. A large fraction of human peripheral
blood gamma/delta+ T cells is activated by Mycobacterium
tuberculosis but not by its 65-kDa heat shock protein. J. Exp.
Med. 171:674.
Modlin, R. L., C. Primez, F.M. Hofman, V. Torigian, K.
Uyemura, T. H. Rea, B. B. Bloom, and M. B. Brenner. 1989.
Lymphocytes bearing antigen specific y8 T-cell receptors accumulate in human infectious disease lesions. Nature 339:
544.
Haregowoin, A., G. Soman, R. C. Holm, and R. W. Finberg.
1989. Human yS+ T cells respond to mycobacterial heat
shock protein. Nature 340:309.
Fisch, P., M. Malkovsky, S. Kovats, E. Sturm, E. Braakmann,
B. S. Klein, S. D. Voss, L. W. Monissey, R. DeMars, W. J.
Welch, R. L. H. Bolhuis, and P. M. Sondel. 1990. Recognition
by human V,9N82 T cells of a GroEL Homolog on Daudi
Burkitt’s lymphoma cells. Science 250:1269.
Fisch, P.,K. Oettel, N. Fudim, J. E. Surfus, M. Malkovsky,
and P. M. Sondel. 1992. MHC-unrestricted cytotoxic and proliferative responses of two distinct human ylS T-cell subsets
to Daudi cells. J. lmmunol. 148:2315.
Singh, B., and R. S. Gupta. 1992. Expression of human 60kDa heat shock protein (HSP60 or P1) in Escherichia coliand
the development and characterization of corresponding monoclonal antibodies. DNA Cell Biol. In press.
Jindal, S., A. K. Dudani, B. Singh, C. B. Harley, and R. S.
Gupta. 1989. Primary structure of a human mitochondrial
protein homologous to the bacterial and plant chaperonins
and to the 65-kilodalton mycobacterial antigen. Mol. Cell.
Biol. 9:2279.
Triebel, F.,F.Faure,M. Graziani, S. Jitsukawa,M. P. LeFranc,
and T. Hercend. 1988. A unique V-J-C rearranged gene encodes a y-protein expressed on the majority of CD3+ T cell
receptor alp- circulating lymphocytes. J. Exp.Med. 167:
694.
Ciccone, E., S. Ferrini, C. Bottino, 0. Viale, I. Prigione, G.
Pantaleo, G. Tambussi, A. Moretta, and L. Moretta. 1988. A
monoclonal antibody for acommon determinant of the human
T cell receptor $8 directly activates CD3+WT31- lymphocytes to express their functional program(s). J. Exp. Med.
168:l.
DeMars, R., C. C. Chang, and R. Rudersdorf. 1983. Dissection of D- region of the human major histocompatibility complex by means of induced mutation in a lymphoblastoid cell
line. Hum. lmmunol. 8:123.
Harlow, E., andD. Lane. 1988. Antibodies:ALaboratory
Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY, p. 47 1.
Voss, S. D., P. M. Sondel, and R. J. Robb. 1992. Characterization of the IL-2 receptors (IL-2R) expressed on human NK
cells activated in-vivo by IL-2: association of the p64 IL-2R
y chain with the IL-2R p chain in functional intermediate
affinity IL-2 receptors. J. Exp. Med. 176:531.
O’Farrell, P. H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250:4007.
Jones, P.P. 1980. Analysis of radiolabelled lymphocyte proteins by one- and two-dimensional polyacrylamide gel electrophoresis. In Selected Methods in Cellular Immunology.B.
B. Mishell and S. M. Shigi, eds. W. H. Freeman & Co., New
York, pp. 398440.
Seong, R. H., C.A. Clayberger,A. M. Krensky, and J. Parnes.
2055
journal of hrnunology
35.
36.
37.
38.
39.
40.
1988. Rescue of Daudi cell HLA expression by transfection
of the mouse beta 2-microglobulin gene. J. Exp. Med. 167:
288.
Demotz, S., H. M. Grey, and A. Sette. 1990. The minimal
number of class I1 MHC-antigen complexes needed for T cell
activation. Science 249:1028.
Harding, C. V., and Unanue, E. R. 1990. Quantitation of
antigen-presenting cell MHC class Iupeptide complexes necessary for T cell stimulation. Narure 346:574.
Schnabl E., H. Stockinger, 0. Majdic, H. Gangitsch, I. J.
Lindley, D. Maurer, A. Hajek-Rosenmayr, and W. Knapp.
1990. Activated human T lymphocytes express MHC class I
heavy chains not associated with beta 2-microglobulin. .I.
Exp. Med. 171:1431.
Allen, H., J. Frazer, D. Flyer, S. Calvin, and R. Flavell. 1986.
Beta 2-microglobulin is not required for cell surface expression of the murine class I histocompatibility antigen H- 2Db
or of a truncated H-2Db. Proc. Natl. Acad.Sci. USA 83:
7447.
Mizzen, L. A., C. Chang, J. I. Carrels, and W. J. Welch. 1989.
Identification, characterization, and purification of two mammalian stress proteins present in mitochondria, grp 75, a
member of the hsp 70 family and hsp 58, a homolog of the
bacterial groEL protein. J. Biol. Chem. 264:20664.
Langer, T., and W. Neupert. 1991. Heat shock proteins hsp60
41.
42.
43.
44.
45.
46.
and hsp70: their roles in folding assembly and membrane
translocation of proteins. Curr. Top. Microbiol.Immunol.
167:3.
Yoshikai, Y., G. Matsuzaki, T. Inoue, and K. Nomoto. 1990.
An increase in number of T cell receptor y/&bearing T cells
in athymic nude mice treated with complete Freund's adjuvants. Immunology 70:61.
F'feffer, K., B. Schoel, N. Plesnila, G. B. Lipford, S. Kromer,
K. Deusch, and H. Wagner. 1992. A lectin-binding, proteaseresistant mycobacterial ligand specifically activates V f l +
human yS T cells. J. Immunol. 148:575.
Rajagopalan, S., T. Zordan, G. C. Tsokos, and S. K. Datta.
1990. Pathogenic anti-DNA autoantibody-inducing T helper
cell lines from patients with active lupus nephritis: Isolation
of CD4-8- T helper cell lines that express the y6 T-cell antigen receptor. Proc. Natl. Acad. Sci. USA 87:7020.
Burdon, R. H. 1986. Heat shock and the heat shock proteins.
Biochem. J. 240:313.
Ferm, M. T., K. Soderstrom, S. Jindal, A. Gronberg, J. Ivanyi,
R. Young, and R. Kiessling. 1991. Induction of human hsp60
expression in monocytic cell lines. Inr. lmmunol. 4:305.
Wand-Wurttenberger, A., B. Schoel, J. Ivanyi, and S. H. E.
Kaufmann. 1991. Surface expression by mononuclear phagocytes of an epitope shared with mycobacterial heat shock
protein 60. Eur. J. Immunol. 21:1089.