Embed Size (px)
Citation preview
ISOLATION AND PARTIAL CHARACTERIZATION OF ANTIGEN-BINDING MOLECULES PRODUCED BY
IN VITRO ‘EDUCATED’ T CELLS*
STEPHEN O’CONNOR,! DIANE EARDLEY,Q FUNC-WIN SHEN? RKHARD K. GERSHONiz and ROBERT E. CONElf
1 Department of Pathology (Immunolo~), Laboratory of Cellular Immunology, Howard Hughes Medical Institute. ZYale University. New Haven. CT 06510. and 3The Sloan Kettering Memorial
Institute, New York, NY 10021, U.S.A.
(Received 27 September 1979)
AMtract-Biosynthetic incorporation of 3H-leucinc or lactoperoxidase-catalysed radioiodination of cell surface proteins was used to radioiabel antigen-specific molecules produced by murine splenic T cells cultured in vitro with heterologous erythrocytes. Antigen-binding proteins present in detergent lysates of labelled cells or in culture media in which labelkd cells were incubated were isolated by specific absorption to and elution from erythrocytes. The eluted proteins were characterized by gei electrophoresis and cohunn chro~to~a~y and were found to bt primarily ~,~72,~ d $ polypeptides, although lower molecular species (35,ObOd, 30,OOOd) believed to bc proteolytic fragments of the larger poiypeptides were sometimes detected. Lyl -r-.2-. Lyl -. 2+. and (by inference) Lyf +.2+ cells produced antigen-binding proteins. However, the majority of cell-associated polypeptides were derived from Ly2 f cells, while antigen-binding proteins secreted by the cells were derived primarily from Lyl+ cells. Moreover, the production of detectable levels of antigen-binding proteins by Ly2+ cells required interaction with Lyl + cells.
lNTRODUC’llON
The molecular basis for T ceil recognition of antigens has been a subject of great interest and of some controversy during the past decade. Early studies which did not use antigen as a concentrating or focusing substrate led to inconsistent results. Immunoglobulin-like molecules have been isolated from T cell membranes which appear to be distinctive from B cell membrane immunoglobulins on structural (Cone & Brown, 1976; Cone et al., X977; Mosely er af., 1979), serological (cone, 1976) and functional (Cone et al., 1974) grounds. On the other hand, several laboratories failed to find any candidate molecules after an exhaustive search (reviewed in Cone, 1977).
Recently, several laboratories have used antigen substrates to concentrate candidate molecules and have found that T cells express
*Supported by Grants CA14216 and A112577, CAIWS. USPHS, National Cancer Institute, Bethesda, Maryiand, U.S.A.
t To whom reprint requests should be addressed. 1 Abbreviations used: -BRBC, burro erythrocytes; BSA,
bovine serum albumin: C’ , comnlement; d, daltons: HuRBC. human erythrocytcs; I& immu~oglobulin; OVA, ovalbumin; RaRBC, rabbit crythrocytes; SDS-PAGE, polyacrylamide gel electrophoresis in sodium dodecyl sulfate; SRBC, sheep erythrocytes.
idiotypic and Ig variable region framework determinant on cell surface mole&es which have antigen binding capacity (Binz t Wigzell, 1976; Eichmann, 1978; Lonai et af., 1978; Rubin et al., 1979). These polypeptides tend to be 150,000 dimers of 68.000-72,000d polypep- tide chains. Immunoglobulin constant region determinants have not been detected on such molecules and the presence of associated light chains is uncertain. The studies of Lonai et al. (1978), however, suggest that light chain variable region framework dete~inan~ are present on LyT2+ T cell receptors. This later result raises the possibility that T cell receptors on LyT subsets may be distinctive.
A second group of T cellderived antigen- specific proteins have been described which are smaller (in contradiction to those described above), bear I-re8ion determinants and have potent immunore8ulatory activity (Tada et al., 1977; Taussig et al., 1975). The ~lations~p between these molecules to the larger polypeptides described above which do not bear I-region determinants (Binz & Wigxell, 1976; Rubin, 1979) remains to be determined,
We have utilized a culture system in which splenic T cells are activated by heterologous erythrocytes to- isolate antigen-binding mol- ecules produced by the activated cells. We have
913
914 STEPHEN O’CONNOR et al.
found these molecules to be generally 68,OOOd polypeptides in the reduced state, but occasionally lower molecular weight species were obser&.AlthoughLyl+,2-,Lyl -,2+ and(by inference) Ly I+ ,2 + ,3 + cells produce these proteins, there was a distinct difference in the amount of antigen-binding material shed or secreted by the cells. The amount of antigen- binding proteins released was reciprocal to the amount found associated with the cells and depended on the Ly phenotype of the producing cell. In addition, while Lyl + cells cultured with erythrocytescouldbeactivatedtoproduceantigcn- binding proteins, Ly2+ cells required interaction with Lyl + cells to become activated for production of these molecules.
MATERIALS AND METHODS
Mice
C57 Bl/6 mice 8-10 weeks of age were obtained from the Jackson Laboratory, Bar Harbor,.ME.
Preparation of T lymphocytes
T cells were purified from whole spleen cell suspensions by passage over nylon wool (Julius et ai., 19731, I~anti-Ig~at~ glass beads (Wig&l, 1976) or non-adherence to goat anti- mouse fg-coated piastic petri dishes (Wysocki & Sato, 1978). The first method provided cell populations containing approximately S-10% Ig-t- cells by immunofluorescence, while the latter two methods yielded populations with less than 1% Ig+ cells.
Production and use of antisera
Anti-Thy 1.2 was obtained from Bionetics, Kensington, MD. Hyb~doma anti-Thy-l sera were obtained as an ascites from Dr. P. Lake or as tissue culture supematant from Dr. J. Sprent. All three antibody preparations killed approximately 50% of normal whole spleen and SO-95% of cultured T cells in the presence of rabbit complement (C’). Congenic anti-Lyl.2 and anti-Ly2.2 were prepared as described previously (Shen et al., 1975). T cells, either before or after culture, were suspended in the appropriate dilution of antisera at 1 x 10’ cells/ml, incubated on ice for 30 min, centrifuged, resuspended in a 1: 10 dilution of rabbit C’, and washed twice before resuspending in media. All antisera were pre-tested for their ability to lyse
appropriate helper or suppressor cells in functional assays.
In vitro stimulation of lymphoid populations by SRBC
Sheep erythrocytes (SRBC) obtained from Colorado Serum Co., Denver, CO were used to, stimulate the appropriate T cell subsets in vitro (Eardley & Gershon. 1976: Eardley et al.. 1979). Briefly, 10’ cells with or without antiserum treatment were incubated with 2 x lo* SRBC for 4 days in Falcon 3008 tissue culture plates under non-rocking conditions in 5% CO,, at 37”C, in modified Mishell-Dutton media (RPMI, 10% fetal calf serum, 1OmM glutamine, 2.5 Hepes, and 5 x 10e5M mercaptoethanol). After 4 days, the T cells were recovered from culture and treated with antisera + C’ as indicated. Dead cells and red cells were removed by ~nt~fu~ng T cells on a layer of lympholyte M (Cedarlane Labs, London, Canada) at 2000 rev/min for 15 min and cells at the interface were collected.
Radiolabelling of cell populations
(a) Incorporation of 3H-ieucine; In vitro activated T cells were separated from RBC and dead cells by centrifugation over LSM. The ceils were then suspended at 10’ cells/ml in leucine- free RPM1 (GIBCG) and l&250 &i 3H- leucine (Amersham, 40-60 Ci~mmole} were added per ml of cells. Cells were allowed to incorporate 3H-leucine for 6 hr at 37°C and were then centrifuged and both cell pellet and supernatant, containing 3H-labelled proteins released during the labelling period, were retained. The cell pellet was lysed with 0.05% T&on-X-100 in Tris-EDTA buffer as described previously (Cone & Brown, 1976) to solubilize cell-associated 3H-labelled proteins. Detergent lysates and the supernatants were dialysed for 6-12 hr against Tris-EDTA buffer which was 0.15M in NaCI, pH 8.0, and then were centrifuged at 10,000 g for 15 min.
(b) Cell surface radioiodination. In vitro activated T cells were labelled with lzsI (New England Nuclear) by lactoperoxidase-catalysed radioiodination as described previously (Emerson et al., 1979). 12JI-Labelled cells were lysed with 0.05% Triton X-100 and the lysates treated as described for 3H-labelled cells.
Antigen binding assay
Dialysed detergent lysates were mixed with an equal volume of buffer to reduce residual detergent below levels which were lytic to
Isolation and Partial Characterization of Antigen-Binding Molecules 915
erythrwytes. Diluted lysate or supematant Cone, 1979). For gels containing * zsI-labelled (OS-l.0 ml) was mixed with 50 jd of a 10% proteins, ~3’I-labelled murine IgM and suspension of erythrocytes and held at 4°C for ovalbumin (OVA) were included as internal 1 hr. The cells are then washed with Tris-EDTA markers. For DATD gels, separate gels buffer at least twice. After washing, the RBC containing 1311-IgM, OVA or bovine albumin were lysed with SDS-PAGE sample buffer and (BSA) and identical amounts of RBC protein an aliquot was mixed with aquasol (New were run to determine the position of molecular England Nuclear) and counted in a Beckman LS- weight standards. The position of standards in 230 Scintillation counter. Xodinated samples gels containing 3H-polypeptides were then were assayed for radioactivity in a Beckman computed by mobility relative to the Biogamma 11 Spectrometer. bromphenol blue dye marker.
Polypeptides bound to the erythrocytes were resolved by discontinuous electrophorcsis in polyacrylamide gels as described previously (Cone & Brown, 1976). except that for 3H- labelled proteins, DATD cross linker was substituted for bisacrylamide. After electro- phoresis, DATD gels were processed for scintillation counting as described (Emerson &
RESULTS
identification of ceil-arsociated and released ~tigen-~i~~g rn~~e~ies
To isolate antigen-binding molecules from sensitized T cells, nylon-purified splenic T cells cultured for 4 days with sheep erythrocytes (SRBC) were centrifuged over Ficoll-Hypaque
SLlCE NO.
0 10 20 w 40 SQ SLICE NO.
Fig. 1. SW-PAGE profile of SRBC-binding proteins isolated from SRBC-sensitized T&is. Nylon puriiied splenic T cells or spleen ceils were cultured for 4 days with SRBC and then labelled for 6 hr with 3H-leucine. S~x~our3H-ieucine-la~lcul~uresu~ma~an~ordc~e~en~ lysattsof”H-labclledceltswtnmixedwithSRBC or BRBC and bound proteins were &ted, reduced and resolved by SDS-PAGE. (A) Proteins in supernatates obtained from SRBC-sensitized T cells bound to SRBC (o-@), BRBC (o-+). (B) Proteins in celi tysates from SRBC-sensitized Tcelis bound to SRBC (o--#, BRBC (O-O). (C) Proteins in supemates obtained from sensitized T celfs 0-O) or spleen cells to-# bound to SRBC. ir, OVA and 2. refer to
mobility of murine Ig p chain. ovalbumin and murine Ig i chain standards.
916 STEPHEN O’CONNOR er al.
and the interface layer ofcells was incubated with 3H-leucine for 6 hr. The 3H-leucine-pulsed cells were then centrifuged and the cell pellet was lysed with Triton X-100. The detergent lysate and the labelling culture supematant which contained released or secreted proteins were dialysed against Tris-EDTA buffer for 6-18 hr. Dialysed, diluted lysates and supernates were then incubated with SRBC or burro erythrocytes (BRBC) for 1 hr at 4°C. The erythrocytes were then washed and lysed with SDS-PAGE sample buffer and 3H-leucine-labelled proteins bound to the cells were reduced and resolved by SDS-PAGE. In several experiments the lysate or supernatant counts/min bound to SRBC were 2 to ‘%-fold higher than those bound to BRBC and amounted to l-3% of the total 3H-leucine counts/min added to the erythrocytes. As shown in Fig. 1, resolution of bound polypeptides by SDS-PAGE revealed a single major peak of radioactivity with an apparent molecular weight of 68,000_72,OOOd. Occasionally minor peaks of activity were observed at 45,OOOd and 30,000-35,OOOd, but polypeptides migrating with immunoglobulin light chains were absent.
The possibility that the 68,OOOd polypeptides might be IgM heavy chains seemed unlikely because of the absence of anti-SRBC PFC in this system (Eardley et al., 1979) and the absence of light chain-sized polypeptides. Nevertheless this point was examined further by (a) clearing supernates of IgM by precipitation with anti-p sera and (b) comparing antigen-binding proteins produced by culture of splenic T cells or spleen cells with erythrocytes. Prior immuno-
precipitation of SRBC-sensitized T cell supematants with anti-IgM sera did not remove the antigen-binding molecules. As shown in Fig.
1 m 3H-leucine-labelled antigen-binding proteins obtained from spleen cell cultures were resolved into peaks of radioactivity which migrated with murine p-chain and light chain standards. In contrast, antigen-binding molecules obtained from splenic T cell cultures were resolved into a major polypeptide chain which migrated slightly faster than p chains and no light chain peak was detected.
Specificity of splenic T ceil antigen-binding molecules for the immunizing antigen
The specificity of the erythrocyte-binding molecules for the immunizing antigen was tested by parallel culture of splenic T cells with sheep erythrocytes, human erythrocytes (HRBC) or rabbit (RaRBC) erythrocytes. Detergent lysates and released (supernates) proteins from jH- leucine-labelled cells were then tested for binding to sheep, rabbit or human erythrocytes. As shown in Table 1, released 3H-labelled polypeptides obtained from sheep or human RBC sensitized T cells exhibited significantly higher binding to the RBC used for sensitization. Similar results were obtained when reciprocal binding assays were done with 3H-leucine- Iabelled proteins in detergent lysates from rabbit or sheep erythrocyte-sensitized T cells. As shown in Figs. 2(A) and (B), resolution of released binding proteins or detergent lysates obtained from human RBC or rabbit RBC-sensitized T cells revealed polypeptides similar to those
Table I. Binding of 3H-leucine-labelled sensitized T cell proteins to antigen”
Exp. Cells cultured with: Tested on: Counts/mm bound Source
SRBC SRBC HuRBC HuRBC SRBC SRBC SRBC SRBC RaRBC RaRBC SRBC SRBC
SRBC 4520 HuRBt’ 2820 HuRBC SRBC SRBC BRBC SRBC RaRBCd RaRBC SRBC SRBC BRBC’
2400 1424
371 1105 550
2815 675
2025 883
Secreted or released Secreted or released Secreted or released Secreted or released Secreted or released Secreted or released Cell associated Cell associated Cell associated Cell associated Cell associated Cell associated
U Nylon puritied splenic T ceils were cultured with RBC for 4 days and then ~dbelied for 6 hr with ‘H-leucine. Detergent lysates of 3H-labelled cells (cell-associated proteins) and supernates containing 3H-Iabelled proteins released or secreted by the cells were then assayed for antigen-binding proteins.
“SRBC. sheep erythrocytes. * HuRBC, human erythrocytes. d RaRBC, rabbit erythrocytes. I* BRBC, burro erythrocytes.
Isolation and Partial Characterization of Antigen-Binding Molecules 917
c OVA ). I
1 1
SLICE NO.
Fig. 2. SDS-PAGE profile of RBC binding proteins isolated from HuRBC or RaRBC-sensitized T cells. Spknic T cells were cultured for 4 days with HuRBC or RaRBC and then IabeIkd for 6 ht with ‘H-kucinc. (A) Reduced, 6 h+H-leucine lobelling supematant derived proteins from HuRBC sensitized ceils which were bound to HuRBC (0-G) or SRBC (@-+. (B) Reduced, cell lysate-derived proteins from RaRBC-
sensitized cells which were bound to RaRBC (o---o~ or SRBC (8-W.
SLICE NO.
POOL NO. Fig 3. Resolution of antigen-binding molecules under reducing or non-reducing conditions. ‘H-leucine- labelkd SRBC binding proteins in 6 hr 3H-leucine label supernates obtained from SRBC-sensitized T cells were resolved by SD!%PAGE under reducing (Panel A) or non-reducing (Panel B) conditions. Supemates were also fractionated by chromatography on Sephadex G-200 (Panel C): (v) chromatographic profile of 3H-leucine-labelIad proteins, (O-O) binding activity of pooled column fractions to SRBC, ( x - x ) BRBC binding activity in pooled column fractions. Blue Dex. IgG and BSA refer to elution point of blue
dextran, murine IgG and bovine serum albumin.
918 STEPHEN O’CONNOR er al.
observed for SRBC-specific polypeptides (Figs. IA and B). A small amount of 68,000_72,00Od proteins was observed binding to iheep erythrocytes which may reflect cross-reactions between SRBC, HRBC and RaRBC. Note that the rabbit RBC specific polypeptides contain an additional minor component migrating with an apparent molecular weight of 28,000d. The origin of these lower molecular weight species, which are occasionally observed, will be discussed further below.
Molecular sizing of non-reduced antigen-binding molecules
To define further the polypeptide composition of the antigen-binding molecules, SRBC specific binding proteins released by 3H-leucine-labelled, SRBC-sensitized T cells were resolved under reducing and non-reducing conditions by SDS-PAGE. As shown in Fig. 3(A), in this experiment one major (72,000) and two minor molecular species (45,000, 30,OOOd) were resolved under reducing conditions. The major molecular species was also present under non- reducing conditions, as was the 30,OOOd component, suggesting that the binding proteins were not composed of covalently linked subunits. To document this point further and to size the binding proteins under nondenaturing conditions, released 3H-leucine-la~lled poly- peptides were fractionated by ,Sephadex G-200 chromatography. Pooled column fractions were then tested for binding to SRBC. Approximately 70% of the labelled polypeptides were collected in the void volume of the column. However, 95?, of the binding proteins were recovered in column fractions between IgG and bovine albumin standards, corresponding to a molecular size of 75,000-80,0006. These results suggest that antigen-binding molecules released by the cells are substantially smaller than conventional immunoglobulins and may in fact be monomeric. However, in some experiments, disulfide bonded dimeric molecules of 140,OOOd have been observed, as well as higher molecular species which may be aggregates. It should be stressed, however, that while molecular species smaller than 72,000--68,OOOd have been detected, we have never observed polypeptides which co- migrated with immunoglobulin light chains.
conventional immunoglobulins. This finding
Source of antigen-binding molecules
The antigen-binding moIecutes described above appear to be distinctive structurally from
Table 2. Effect of preculture treatment of splenic T cells on the detection of antigen-binding proteins“
Antiserum treatment
rtyi +c’ xLy2 +c’
Cells cultured
Lyl -,s+ Lyl+.2-
Supematant (countsjmin bound)
SRBC HuRBC
3905 3689 25,850 3305
1Ly2 -K’ Lyl+.2- =Ly l+c’ Lyl-.2-l-
+ 60.610 6252
“Nylon purified splenic T cells were treated with anti-Ly sera+C’ before culture with SRBC. After culture the cells were labelled with JH-leucine, centrifuged and supernates containing released 3H-labelled proteins were tested for antigen-binding proteins.
makes it unlikely that such molecules were produced by the few B lymphocytes or plasma cells that might be present after 4 days culture. Moreover, similar molecules have been isolated from T cell cultures in which the T cells were prepared by either passage down anti-mu&e Ig columns or by negative selection on anti-Ig plates. In an effort to define the T cell source of the antigen-binding molecules further and delineate which T cell subsets might produce these molecules, T cells were treated with anti- LyT sera and complement either before culture with SRBC or after culture but before labelling with 3H-leucine. As shown in Table 2, Lyl+,2 - cells or combined populations of Lyl + ,2 - and Lyl - .2 + cells cultured with SRBC synthesized and released antigen-binding molecules. No
SLICE NO.
Fig. 4. SDS-PAGE profile of antigen-binding proteins produced by anti-Ly2 + C’-treated splenic T cells. Splenic T cells were treated with anti-Ly2 sera+C’ and then cultured For 4days with SRBC. Thecells were labelied with 3H-leucine and supernates and detergent lysates were assayed for antigen-binding proteins. Bound proteins were reduced and resolved by SDS-PAGE. (+--8) Lysate proteins bound to SRBC, (Q--O) supernatant proteins bound to SRBC. (A-A) lysate proteins bound to BRBC. f x-x 1
sunernatant binding proteins bound to BRBC. _
Tab
le
3.
Eff
ect
of
anti
-Ly
sera
an
d C
’ tr
eatm
ent
of
cult
ure
d T
cel
ls o
n t
he
det
ecti
on
of
anti
gen
-bin
din
g p
rote
ins“
L
x
Mo
lecu
iar
spec
ies
det
ecte
d
$
An
tise
rum
2.
S
ou
rce
of
68,o
oo
35
,000
30
,080
0,
trea
tmen
t b
ind
ing
pro
tein
s co
un
ts/m
in
:“, t
ota
l co
un
tJm
in
:‘:,
tota
l co
un
ts/m
in
‘% to
tal
To
tal
~ou
nts
/min
re
du
ctio
n (
‘J&
) 8
NM
S+C
L
ysat
e 13
13
41
1021
32
84
2 Ij
An
ti-L
yl
+C
3176
L
ysat
e 78
.5
57
238
I7
360
::
An
ti-L
yZ+C
13
83
56
2.
Lys
ate
275
39
212
30
223
31
An
ti-L
yl
+an
ti-L
y2+C
’ 71
0 78
lp
L
ysat
e 13
0 82
29
18
0
c
NM
S+C
I5
9 95
S
up
emat
ant
500
B
80
- 12
: 20
62
8 aL
yl
+c
Su
pem
atan
t
-
IO0
0 -
- -
aLyZ
+C
Su
per
nat
ant
-
::
74
- -
15
26
:z
::
z f.
“C57
B1/
6sp
ien
icT
cells
w
ere
pre
par
ed b
y d
ep
letio
n o
f B
cells
on
an
ti-1
8 p
late
san
d w
erec
ult
ure
d f
ot4
day
s w
ith
SR
BC
or
BR
BC
. T
hec
ells
wer
e th
en t
reat
ed w
ith
NM
S
or
anti
Ly
T s
eraa
nd
C
b
and
wer
e th
en p
uls
ed fo
r 6
hr
wit
h J
H-f
euci
ne.
Six
-ho
urc
ult
ure
sup
ern
atan
ts
and
0.0
5’:”
Tri
ton
X-1
00 l
ysat
es o
f JH
-lab
elle
d c
ells
wer
e m
ixed
wit
h S
RB
C o
r B
RB
C.
Po
lyp
epti
des
bo
un
d b
y th
e R
BC
5
wer
e cl
ute
d w
ith
SD
S-P
AG
E
sam
pie
bu
ffer
, re
du
ced
an
d r
eso
lved
by
SD
A-P
AC
E.
Dat
a re
pre
sen
ts th
e in
teg
rate
d c
ou
nts
jmin
fr
om
gel
pea
ks r
eso
lved
. Co
un
ts/m
in
bo
un
d t
o B
RB
C
wer
e 2
ind
isti
ng
uis
hab
le f
rom
det
ecto
r b
ackg
rou
nd
fo
r ea
ch s
amp
le.
c 3
920 STEPHEN O’CONNOR et af.
binding proteins were detected when Lyl - .2 + cells were cultured with SRBC. The SDS-PAGE profile of reduced and alkylated binding proteins recovered from supernates or cell lysates obtained from jH-labelied LyI +.2 - ceils which had been cultured with SRBC were essentially similar to profiles obtained from unseparated T cells (Fig. 4) with 68,000d polypeptides comprising the major molecular species. It should be noted, however, that cell Iysates obtained from Ly 1 + .2 - cells contain a 48,000d binding protein which was not detected in polypeptides released by the cells.
The results presented above suggest that antigen-binding proteins may be obtained when Ly 1 f.2 - cells alone are cultured with antigen Lyl + ,2- cells might either be the sole source of such proteins or Ly2+ cells may require interaction with Lyl + cells to become activated. Consequently, splenic T cells were cultured with SRBC and after 4 days cultures were treated with anti-Thy-l or anti-Ly sera and C’ before labelling with 3H-leucine. Anti-Thy-l treatment reduced the amount of labelled antigen-binding proteins detected in cell lysates by 83x, but had no effect on the quantity of antigen-binding molecules detected as released molecules. Similar results were obtained when anti-Thy-l-coated cells were mixed with a rabbit IgM anti-mouse Ig serum + C’.
The effects of anti-Ly sera and C’ treatment on the recovery of antigen-binding molecules was assessed by analysis and integration of the SDS-PAGE profiles of the binding proteins detected (Table 3). In this way possible variations in molecular species of binding proteins with LyT subsets could be determined. Three molecular species of antigen-binding molecules (68,000, 35,000 and 30,OOOd) were detected in cell lysates of NMS-treated cells. Treatment of the cells with anti-Ly 1 andanti-Ly2 sera and C’ resulted in loss of 95% of the antigen- binding proteins. However, treatment of the cells with anti-Lyl or Ly2 sera resulted in differential recovery of antigen-binding molecules from cell lysates or supernates. Seventy-eight per cent of the antigen-binding molecules in the cell lysate were lost by removal of Ly2 i- cells, whereas 56% were lost by removal of Lyl + cells. The SDS-PAGE profiles of Ly I+ ,2 - or Ly 1 - ,2 + cells were essentially identical except that a greater proportion of the binding proteins in the Lyl + ,2 - set were 35,000d. In contrast, 75% of the antigen-binding molecules in the supernates were lost by treatment of the cells with anti-Lyl
Table 4. Contribution of antigen-binding proteins by LyT subsets to cell lysates and supernatants”
Per cent of binding protein in Lysate Superoatant
Ly phenotype (cell-associated) (released)
Lyl+.2- 22 45 Lyl+.2+ 34 30 Lyl-.2f 44 25
“Calculations were made from data presented in Table 3.
sera and 55% were lost by treatment with anti- Ly2. Note also that in the supernates 75-100x of the binding proteins were 68,000d. Based on the percentage loss of binding proteins achieved with tr~tment of the cells with anti-Ly sera, we calculated the contribution of binding proteins in cell lysates and supemates (Table 4) by distinct LyT phenotypes. Twenty-two per cent of the antigen-binding proteins in the cell lysate were contributed by Lyl+,2- cells, 34% by Lyl + ,2 + cells and 44% by Lyl - ,2 + cells. In contrast, 45% of the binding proteins in supernates were contributed by Lyl + ,2 - cells, 30% by Lyl + ,2+ cells and only 25% by Lyl-,2-l- cells.
The results described above indicate that splenic T cells sensitized in vitro to erythrocytes synthesize and release polypeptides with binding specificity for the immunizing antigen. These molecules may be derived from the surface membrane or could be secreted analogs of a membrane receptor. To ascertain the nature of surface-associated antigen-specific molecules, splenic T cells cultured in vitro with SRBC were radiola~Iled by la~to~~xidase-cataly~d radioiodination. Radiolabelled cells were lysed with detergent and antigen-binding molecules were detected by incubation of detergent lysates with SRBC or BRBC. Labelled polypeptides bound to the erythrocytes were eluted, reduced and resolved by SDS-PAGE. SRBC bound 2.1 x 1 O4 counts/min and BRBC bound 1 x IO4 counts/min after 3 washes. SDS-PAGE of polypeptides bound to BRBC did not reveal significant polypeptide peaks, whereas poly- peptides bound to SRBC were resolved into a major peak of radioactivity with an apparent molecular weight of 68,000d (Fig. 5). The surface radiolabelled antigen-binding molecnles thus resembled those detected as cell-associated or released molecules obtained from “H-leucine labelled cells.
Isolation and Partial Characterization of Antigen-Binding Molecules 921
2 0 IO 20 30 40 SO a
SLICE NO.
OVA
1
Fig. 5. SDS-PAGE of surface labelled proteins of antigen binding proteins. Splcnic T cells were cultured for 4 days with SRBC and then surface radiolabelfed with ts51 by lactoperoxidase-catalysed radioiodination. The cells were lysed with 0.05% Triton-X-100 and the detergent lysate mixed with SRBC or BRBC. Proteins bound to SRBC or BRBC were elutcd, reduced and resolved by SDS-PAGE. Eluates from BRBC did not show any peaks of activity above
detector background.
DISCUSSION
In this study we have shown that murine splenic T cells cultured in vitro with heterologous erythrocytes produce proteins which exhibit binding specificity for the sensitizing antigen. Since the specificity of these binding proteins parallels the functional (Eardley % Gershon, 1976) and antigen binding capacity (Eardley et al., 1979) of T cells induced in this culture system, it is likely that these molecules were produced by cell populations which had undergone clonal expansion as the result of antigenic stimulation.
Reduced and alkylated antigen-binding molecules were primarily 72,000-68,000<1 polypeptides, although in some cases lower molecular weight species of 35,000, 30,000 and 18,000d were observed. Since the smaller polypeptides were also observed under non- reducing but denaturing conditions, they do not appear to be covalently linked by disulfide bonds to the 68,OOOd chains, although non-covalent linkages cannot be ruled out.
However, the lower molecular weight species could represent proteolytic cleavage products of the 68,000d polypeptide chain. The observation that these molecules are not always observed, and tend to be associated more with cell lysates than with antigen-binding proteins released into
the culture medium during labelling supports this contention. Marked sensitivity of T cell receptors to proteolysis has been described (dinz & Wigzell, 1976) with cleavage products similar in size to the lower molecular weight molecules
observed herein. In addition, 68,OOOd T cell suppressor factors with specificity for TNP or DNP show marked sensitivity to proteolysis (Cone et al., 1979), which is particularly evident when these molecules are aggregated either by binding to antigen or when they are bound by antisuppressor factor antibody. The major cleavage products in this case are 45,000,35,000 and 25,OOOd. A portion of these lower molecular weight species have antigen binding capacity.
Analysis of the culture medium derived antigen-binding molecules under non-reducing. denaturing or non-denaturing conditions indicated that most of the molecules were in a monomeric form. However. in some experi- ments, dimers of 150,OOOd were also detected. These observations’ are consistent with previously described characteristics of T cell proteins which bind antigen and/or bear idiotypic determinants (Taussig et al., 1979; Binz & Wigzell, 1976; Rubin et al., 1979). The fact that both monomeric and dimeric antigen-binding molecules have been observed may indicate that more than one form of T cell antigen recognition molecule exists. Conceivably, secreted molecules might tend to be monomeric while membrane receptors are dimeric. Alternatively, the properties of these molecules may be such that artifacts are generated during isolation. Thus, the molecules may normally exist as monomers but when they are released by cells or are extracted with detergent they may aggregate and inter-chain disulfide bonds may be formed by disulfide interchange. Conversely, proteolytic cleavage above or below interchain disuhide bonds could create molecules which appear to be composed of non-covalently linked polypeptide chains.
Since both membrane bound and intracellular proteins are labelled by incorporation of 3H- leucine, the subcellular origin of the 3H-labelled antigen-binding molecules cannot be deter- mined. However, antigen-binding molecules with similar molecular characteristics to the endogenously labelled proteins were isolated from surface radiolabelled, SRBC-sensitized cells. The SDS-PAGE pattern of surface labelled antigen-binding proteins identified in this study is very similar to that obtained from in viva activated SRBC-specific T cells (Cone & Marchalonis. 1973). Whether membrane- associated antigen recognition structures are identical to those released by T cells remains to be determined.
The structure of antigen-binding molecules
922 STEPHEN O’CONNOR et ol.
produced by T cells sensitized in vitro suggests that these polypeptides are not conventional immunoglobulin molecules produced by B cells or plasma cells. Although the ‘heavy’ polypeptide chains produced by T cell cultures are similar in size to IgM heavy chains, precipitation of supemates with anti-p sera did not remove the antigen-binding proteins. Moreover, antigen-binding proteins isolated from culture fluids obtained from in vitro sensitized spleen cells possessed polypeptide chains which migrated with immunoglobulin light chains, while splenic T cell supernates contained only 68,000d polypeptides. Our inability to detect light chain-sized molecules associated with the antigen-binding T cell products suggests either that these molecules do not possess light chains or that the light chains may be noncovalently bound to heavy chains but are lost during binding to antigen. Preliminary evidence, however, suggests that the antigen-binding proteins bear murine Ig variable region framework determinants and idiotypic determinants associated with murine anti-SRBC antibody.
Post culture treatment of the cultured cells with anti-Ly sera establishes clearly the source of the antigen-binding molecules as T cell-derived. The striking finding in these studies is the relative contribution of antigen-binding molecules to cell lysates or supernates by LyT subsets. Most of the antigen-binding molecules in the cell lysates were derived from Ly2 + cells; the greatest proportion produced by Lyl - ,2+ cells. In other experiments the proportion shifted somewhat to Lyl + ,2+ cells, but in no case did Lyl+,2 - contribute more than 25% of the antigen-binding molecules in the lysate.
In contrast to the results obtained with cell lysates, 75% of the antigen-binding molecules released by the cells into the culture medium during labelling were derived from Ly 1 + cells, and 60% of this material was produced by Lyl + ,2- cells. These results suggest that Ly 1 + ,2 - cells may be primarily ‘secretory’ cells which rapidly release antigen-binding molecules. This may explain the difficulty in detecting Lyl+.2-RFC (Eardley ei a/., 1979) and the observation that only a small proportion of antigen-binding molecules in the cell lysate are contributed by these cells. Lyl - ,2 + cells may thus be non-secreting cells (which bind antigen) under these conditions. However, it is possible that some Lyl - ,2+ cells can be induced to secrete antigen-binding molecules by interaction
with Lyl + cells. Lyl+,2+ cells appear to be equally capable of both antigen binding (Eardley et al., 1979) and release of antigen-binding molecules, although whether the same cell can perform both functions remains to be determined. It should be stressed that the Ly phenotype of antigen-binding cells is critically dependent on both antigen dose and the period after exposure to antigen at which the cells are assayed (Eardley ef al., 1979). It may well be that a similar situation obtains for the subset source of cell-associated or released antigen-binding molecules.
The sensitivity of cells producing antigen- binding molecules to treatment with anti-Ly sera and C’ provides strong evidence that such molecules are produced by T cells. The observation that postculture treatment of the cells with anti-Thy 1 sera and C’ removes most of the source of the antigen-binding molecules provides further support to this contention. However, anti-Thy-l treatment was not as effective in depleting lysate-derived material as anti-Ly treatment. Thus, while the majority of cells which produce antigen-binding molecules detected to cell lysates were eliminated by anti- Thy-l treatment, none of the cells releasing antigen-binding molecules during culture are sensitive to anti-Thy-l antibody and C’. That such ceils are indeed T cells is shown by their sensitivity to anti-LyT sera and C’. The resistance of cultured T cells to lysis by anti-Thy- 1 sera and C’ has been reported elsewhere (Eardley & Gershon. 1976; Eardley er al., 1979). Conceivably the Thy-l-resistant suppressor cells described previously (Eardley & Gershon, 1976) may be sensitized T cells which are secretiug antigen-binding molecules with immunobiologic activity.
Removal of Lyl + or Ly2+ T cells before culture with erythrocytes revealed that antigen- binding molecules could be produced by Ly2- depleted populations, whereas no molecules were produced by Lyl-depleted populations. Since Ly 1 - ,2 + cells cultured in the presence of Lyl + cells produce antigen-binding proteins (see above), the results suggest that Lyl - ,2+ cells require interaction with Lyl + cells to become activated (Eardley et al., 1979) and/or are derived from Ly 1 + ,2 + precursor cells. The effects of removal of LyT subsets on the subsequent activation and production of antigen-binding molecules by the remaining cells differs from results obtained previously (Eardley et al.. 1979) when the effect on the generation of
Isolation and Partial Characterization of Antigen-Binding Molecules 92.1
antigen-binding (rosette-forming) cells was assessed. In those studies T-RFC were not generated when the cells were pretreated with either anti-Lyl or anti-Ly2 sera and c’. In the present study it is clear that T cells can become activated to produce and secrete antigen-binding molecules under conditions in which no rosette- forming cells can be found.
A consistent picture is beginning to emerge with respect to the molecular characteristics of T cell products which bind antigen specifically. Based on the results of our study as well as those of others (Binz & Wig&l, 1976; Taussig et al., 1979; Rubin et al., 1979), it has been shown that antigen-binding T cell proteins bear idiotypic determinants expressed on serum immuno- globulins, suggesting that the specificity of the T cell products is coded for by immunoglobulin variable region structural genes. In no case, however, have immunoglobulin constant region dete~inants been shown de~nitively to be associated with these molecules. Whether all T cell antigen recognition structures carry light chains remains problematic. Antigen-binding proteins isolated by their affinity for antigen or anti-idiotype antibodies in most cases do not have light chains. Light chains non-covalcntly linked to heavy chains could be lost when the combining site is perturbed by anti-idiotype or antigen. Antisera to immunoglobulin light chain variable region framework determinants have been reported to inhibit antigen binding by some T cell subsets (Lonai ef al., 1978). Some mammalian (reviewed in Cone, 1977) and avian (Szenberg et a/.. 1977: Warr ef al.. 1978) antisera to Fab determinants bind to solubilized T cell membrane proteins (IgT) which appear to possess light chain-sized poly~ptides. We have found, however, that the light chain-sized polypeptides present when labelled T cell membrane proteins are bound by anti-Fab antibodies are not antigenically identical to serum Ig light chains (Cone & Rosenstein, unpublished results). The antibodies responsible for binding T cell membrane proteins appear to be specific for immunoglobulin variable region framework determinants. Thus, if some T cell antigen recognition molecules possess light chains, they may well represent a new light chain isotype. On the other hand, light chain-sized molecules can be generated by spontaneous proteolysis of hapten affinity purified, antigen- specific 68,000d T cell suppressor factors or by immunoprecipitation of these factors by anti- suppressor factor serum (Cone et al., 1979).
Thus, the demonstration of light chain-sized polypeptides cannot by itself be taken as evidence for the existence of light chains associated with T cell antigen r~ognition molecules.
Definition of the molecular basis of T cell recognition of antigen is complicated further by the description of immunobiologically active, antigen-specific molecules which bear I-region determinants. These molecules are generally smaller than those described above, but in some instances have been shown to carry immunoglobulin variable region determinants (Greene et al., 1979). Since most of the 68,OOOd species described above do not bear I-region determinants (Binz & Wigzell, 1976; Rubin er of., 1979), i-region bearing antigen recognition molecules may either be a second kind of antigen recognition structure or such factors are formed by a post-synthetic association of fragments of an antigen-binding molecule with I-region gene products. Evidence in favor of this contention has been reported by Taussig et al. (1979).
The last five years has seen the T lymphocyte lineage subdivided into multiple interacting subsets distinguished by their cell surface phenotype of MHC and diff~tation antigens and their function. The common (?) denominator for these subsets appears to be antigen recognition molecules which, based on the results of this study, are similar in structure. It is clear, however, that LyT subsets may be differentiated based on whether their antigen recognition molecules can be released or secreted by the cell. Lyl + ,2 - cells tend to secrete antigen recognition molecules, while Lyl - ,2+ cells cultured alone do not secrete antigen recognition molecules. Now this observation relates to the function of these subsets remains to be determined. In addition, although Lyl*+ and Ly2+ antigen recognition molecules appear to be similar structurally, it is possible that they are different isotypically. Possible isotypic differ- ences may also relate to both receptor function and the immunobiological activity of soluble antigen recognition molecules. The detailed characterization of these molecules is thus likely to provide insight into the differentiated function of T lymphocyte subsets,
REFERENCES
Binz H. & Wig&l H. (1976) Shared idiotypic determinants on B and T lymphocytes reactive against the same
924 STEPHEN O’CONNOR er al.
antigenic determinants. V. Biochemical and serological characteristics of naturally occurring soluble antigen binding molecule. &and. J. Immun. 5, 559-571.
Cone R. E. (1976) Factors influencing the isolation of membrane immunogiobulins from T and B lymphocytes. 1. Detergent effects and iodination conditions. J. Immun. 116,847-853.
Cone R. E. (1977) The search for the T cell receptor. Prog. Immun. 3,47-57.
Cone R. E. & Brown W. (1976) Isolation of membrane- associated immunoglobulins from T lymphocytes by non- ionic detergents. Immunochemisrry 13, 571-579.
Cone R. E., Feldmann M., Marchalonis J. J. & Nossal G. J. V. (1974) Cytophilic properties of surface immunogiobulin of thymus-derived lymphocytes. immunology 26, 49-60.
Cone R. E. & Marchalonis J. J. (1973) Antigen binding specificity of cell surface immunogiobuhn isolated from T (helper) cells. AU?. J. exp. Eiol. med. Sci. 51, 689-700.
Cone R. E., Hoe&i D. & Rosenstein R. W. (1977) Analysis of detergent-extracted membrane immunoglobulins of T and B lymphocytes by ultracentrifugation and column chromatography. Immunochemistry 14, 345-352.
Cone R. E., Rosenstein R. W., Kondo K., Murray J., Ptak V. & Gershon R. K. (1979) Characterization of T cell-derived molecules involved in antigen recognition. J. supramolec. Struct. Suppl. 3,~. 256 (Abs.).
Eardley D. D. & Gershon R. K. (1976) Induction of specific suppressor T cells in vitro. 1. Immun. 117, 313-318.
Eardley D. D., Shen F. W., Cone R. E. 8c Gershon R. K. (1979) Antigen-binding T cells: Dose response and kinetic studies on the development of different subsets. J. Immun. 122 140-145.
Eichmann K. (1978) Expression and function of idiotypes on lymphocytes. Adv. It&m. 26, 195-254.
Emerson S. G. & Cone R. E. (1979) Turnover and sheddina of Ia antigens by murine spleen cells in culture. J. Immun. 122,892-899.
Warr G. W., Marton G., Szenberg A. & Marchalonis J. J. (1978) Reactions of chicken antibodies with immunoglobuhns of mouse serum and T cells. Immunochemistry 15, 615-622.
Emerson S. G., Reilly P. & Cone R. E. (1979) Differential Wigzell H. (1976) Enrichment or depletion of surface Ig-
radiolabelhng of lymphocyte membrane alloantigens and coated cells using anti-Ig antibodies and glass or plastic immunoglobulins: Variation of H,O, concentration bead columns. In In vitro Methods in Cell-mediated and during lactoperoxidasecatalyd cell surface radio- Tumor Immunity. pp. 245-25 I. Academic Press. New York. iodination. 1. Immunogener. 6, 87-97. Wysocki L. J. & Sato V. L. (1978) Panning for lymphocytes: a
Greene M. I., Bach B. A., Nisonoff A. & Benacerraf B. (1979) method for cell selection. Proc. nam. Acad. Sci. U.S.A. 75, VH gene products are present on suppressor T cell-derived 284+2848.
regulatory molecules. J. supramolec. Struct. Suppl. 3 p. 256 (Abs.).
Julius M. H., Simpson E. & Hmenberg L. A. (1973) A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur. 1. Immun. 3, 645-651.
Lonai P. Ben-Neriah Y., Steinman L. & Givol D. (1978) Selective participation of immunoglobulin V region and major histocompatibility complex products in antigen binding by T cells. Eur. J. Immun. 8, 827-832.
Mosely J. M., Beatty E. A. & Marchalonis J. J. (1979) Molecular properties of T-lymphoma immunoglobulin. 1. Immunogenet. 6, l-18.
Rubin B., Hentel-Wulff B. 8~ Kimura A. K. (1979) Characterization of alloantigen-specific T cell receptors. J. supramolec. S/rtrct. Suppl. 3. p. 256 (Abs.).
Shen F. W., Boyse E. A. & Cantor H. (1975) Preparation and use of Ly antisera. Immu.nogenerics 2, 591-595.
Szenberg A., Marchalonis J. J. & Warner N. L. (1977) Direct demonstration of endogenous murine T cell surface immunoglobulin. Proc. nom. Acad. Sci. U.S.A. 74, 2113-2118.
Tada T., Taniguchi M. & David C. S. (1977) Suppressive and enhancing. T cell factors as I-region gene products: properties and the subregion assignment. Cold Spring Harb. Symp. quant. Bioi. 41, 119-128.
Taussig M. J., Munro A. J., Campbell R., David C. S. & Staines N. A. (1975) Antigen-specific T cell factor in ceil cooperation. Mapping within the I-region of the H-2 complex and ability to cooperate across allogenic barriers. J. exp. Med. 142,694-700.
Taussig M. J. & Holliman A. (1979) Structure of an antigen- specific suppressor factor produced by a hybrid T cell line. Nature, Lond. 277, 308-310.
