- Email: [email protected]
Cell interactions in the immune response in vitro,
CELLULAR
IMMUNOLOGY
Cell
9,
l-11
(1973)
Interactions
in the Immune
VI. Mediation
by T Cell Surface
P\/IAKc
Walter
J.
MARCIIALONIS~
Iustitute Hospital,
of Medical Melbourne,
JOHN
and E&a Hall Royal Melbourne
Monomeric
Received
IgM1
ROBERT E. CONE,~
FIXDMANN,” AND
Response in Vitro
August
Research, Post 3050, Australia
Ofice,
11, 1972
The nature of the specific mediator of T/B lymphocyte cooperation was investigated by using biochemical and in vitro culture methods in parallel. By surface radioiodination of activated thymus cells it was found that the T cell component of cell interaction was an immunoglobulin which possessed a mobility on polyacrylamide gel electrophoresis consistent with a molecular weight of approximately 180,000. This mobility of molecule comprised of two polypeptide chains, with the electrophoretic light and p chains. This monomeric IgM molecule had specificity for the activating antigen, and like the mediator of cell interaction, bound cytophilically to macrophages, but not to T or B lymphocytes. These studies extend previous work in which the mediator of cell cooperation was found, by the use of antisera to contain antigen, and K and p chain determinants. The binding of the T cell surface IgM to macrophages supports the concept that the final step of cell cooperation, the immunization of B cells occurs at the surface of macrophages, occurs via a lattice of antigenic determinants created by cytophilic T cell carrier antibody.
INTRODUCTION Collaboration between thymus derived (T) and non thymus derived (B) lymphocytes is required for optimal antibody responses to many antigens, such as heterologous erythrocytes or hapten protein conjugates (1-5). The mechanisms by which T cells facilitate antibody production by B cells has recently been investigated in vitro. It was found that direct contact of T and B lymphocytes was not essential (6, 7)) but that active T cell metabolism was required (8). These results suggested that lymphoid cell collaboration was mediated by a released subcellular T cell component. Furthermore, it was found that adherent cells presumably macrophages were required for the production of antibody in vitro to thymus dependent antigens, even to those of small size, whereas adherent cells were not essential for antibody 1 This work was supported by grants from the National Health and Medical Research Council, Canberra, Australia, the Australian Research Grants Committee, the National Institute of Allergy and Infectious Disease (AI-o-3958) and the American Heart Association (721050). This is publicatoin No. 1720 from the Walter and Eliza Hall Institute. a Present address : Tumour Immunology Unit, Department of Zoology, University College London, England. * Postdoctoral fellow of the Damon Runyon Memorial Fund for Cancer Research. l Address requests for reprints to: J. J. Marchalonis, Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Melbourne, 3050, Australia.
Copyright All rights
0 1973 by Academic Press, of reproduction in any form
Inc. reserved.
2
FELDMANN,
CONE,
AND
MARCHALONIS
production to thymus independent antigens (9). This observation suggested that macrophages or macrophage-like cells are involved in lymphocyte cooperation. This was confirmed by culturing these cells, separated by a cell impermeable nucleopore membrane, from antigen activated thymus cells (ATC) and antigen. In this system it was shown that the macrophages had acquired the capacity to induce antibody formation in B cell populations selectively to the antigen used to activate the T cells (10). Because the macrophages only acquired immunogenic capacity in the presence of specific ATC, it appeared that the macrophages were influenced by a T cell subcellular component (10). Trypsinization of the macrophages which had been cultured with ATC and antigen demonstrated that the immunogenic moiety was present on the cell surface. Serologic studies using anti-antigen and antiimmunoglobulin heavy and light chain antisera indicated that the specific mediator of cell collaboration was a complex of T ccl1 IgM and antigen (10). Lactoperoxidase-catalysed radioiodination (11) of cell surface proteins (12-14) has enabled the isolation of cell surface immunoglobulin from both T (15, 16) and B (14, 16, 17) lymphocytes. T cell surface immunoglobulin is a monomeric IgM molecule (15, 16, 18) which, when obtained from activated cells possesses binding specificity for the activating antigen (18). Thus it seemed probable that the T cell IgM involved in cell collaboration was similar to the T cell receptor for antigen. In the present communication, parallel immunological and biochemical evidence is presented which identifies the T cell component of the collaborative factor as a monomeric IgM molecule. MATERIALS
AND
METHODS
Aniwds CBA/H/Wehi mice were used for most experiments. Congenitally athymic (nu/nu) mice provided a source of lymphoid cells devoid of T lymphocytes Activation
of Thyrnus
nude (19).
Cells
Lethally irradiated (800 rad) mice were injected with syngeneic thymocytes and antigen, as described elsewhere (8). Six to seven days later their spleens served as a source of activated thymus cells (ATC) . Antigens Keyhole limpet hemocyanin (KLH) was obtained from Calbiochem, Sydney, Australia. Fowl gammaglobulin (FyG) was prepared as described by Miller and Warner (20). The dinitrophenyl (DNP) determinant was conjugated to KLH, FyG, and flagella of Salmonella adelaide (Fla) as described elsewhere (21) . Inwnunization CBA mice were injected intraperitoneally were used in culture l-3 months later. Tissue
with
25 pg DNP
Fla. Their
spleens
Culture
Mouse spleen cells were cultured in a modified Marbrook-Diener For routine generation of responses, single compartment culture
culture system. flasks were used.
CELL
INTERACTIONS
In
Vitro
BY
T
CELL
IgM
3
Cultures with 2 compartments were performed as described elsewhere (7). The two compartments were separated by a cell impermeable nuclepore membrane of 0.2 0 pore size (Nuclepore, General Electric Co., U.S.A.). ATC were placed in the upper compartment and various cell populations in the lower chamber. Media and sera used for culture were as in previous communications (7) _ Enumeration
of Antibody
Forming
Cells (AFC)
Cells forming antibody were enumerated by the Cunningham and Szenberg fication (22) of the hemolytic plaque assay, as described elsewhere (21). Peritoneal
Exudate
Six to eight months proteose peptone broth 4-5 days later. Ma.crophage
modi-
old CBA mice were injected (Difco, Michigan). Peritoneal
intraperitoneally with 1 ml exudate cells were obtained
Depletion
The active adherence column technique of Shortman et al. (23) was used. Basically, spleen cells suspended in 50% mouse serum are passed rapidly through a column of large siliconized glass beads at 37°C. The effluent is extensively depleted of phagocytes (23). Antisera Rabbit antiserum to KLH was prepared by immunizing a rabbit twice with 1 mg KLH emulsified in Freund’s complete adjuvant. Rabbit anti fowl gamma globulin antiserum was generously provided by Dr. B. T. Rouse. This antiserum bound to IgM and to the 7s immunoglobulin of the chicken. Preparation
of Cell Surface
Imwtunoglobulin
Activated T cells were sedimented through bovine serumatin (BSA) gradients (24) to remove dead cells and debris. The cells were then washed 5 times in phosphate-buffered saline, pH 7.2 and iodinated to high specific activity using lactoperoxidase catalyzed radioiodination (12). Briefly, lo7 cells were suspended in 50 ~1 lactoperoxidase (25 pg) and 200 &i W (iodide) and 10 ~1 of 0.03% H202 were added. The reaction was initiated by vigourous shaking and the cells washed twice with PBS. Fifty to 100 million cells were iodinated in aliquots of 1 x 10’ cells. After iodination the cells were incubated Lmder short-term cell culture conditions for 3 hr, centrifuged and the supernatants retained as described previously (15, 16). Under these conditions, cell surface proteins, including immunoglobulin are released into the cell culture medium (16, 2.5). Radioactive cell surface immunoglobulin was isolated from the supernatants by specific coprecipitation using rabbit anti mouse immunoglobulin antiserum and purified mouse G immunoglobulin as carrier (15, 16). The rabbit antiserum reacted with light chains and p heavy chains. Radioactivity was determined in a Packard Autogamma scintillation counter with a deep well Nal crystal detector.
FELDMANN,
4
CONE,
AND
MARCHALONIS
FIG. 1. Analysis by polyacrylamide gel electrophoresis in acid urea of specifically coprecipitated surface immunoglobulin from T cells activated to KLH. Intact immunoglobulins resolved on 0, surface immunoglobulin from activated T cells; O- - - -0, specifically 4% gel. 0 coprecipitated mouse rG immunoglobulin. Under those conditions 19s IgM does not enter the gel.
Resolution (PAGE)
of Cell Surface
Iwwnunoglobulin
by Polyacrylanzide
Gel Electrophoresis
The molecular size of intact cell surface immunoglobulin was determined by dissolving coprecipitated immunoglobulin in 9 M urea, 10% acetic acid. Coprecipitated immunoglobulins were also dissolved in 9-M urea and reduced and alkylated to cleave interchain disulfide bonds (26). Intact and reduced and alkylated samples and immunoglobulin markers were resolved by disc electrophoresis in acid urea (27). Cell and Antigen
Binding
Assays
z&h
Cell Surface
Proteins
The ability of ATC cell surface proteins to bind to various cell types was determined by adding 100 ~1 of radioactive cell surface proteins to 2 X lo6 CBA peritoneal macrophages, CBA thymus cells or nu/nu spleen cells in 100 ~1 phosphate buffered saline (PBS, pH 7.2) containing 10% FCS. The cells were incubated for 1 hr at 4°C and washed 46 times with PBS containing 10% FCS. Radioactivity was monitored in the pellet after each wash. The antigen binding specificity of ATC cell surface proteins was determined by coprecipitation with the activating antigen and a rabbit antiserum to the antigen. Briefly, 100 ~1 of radioactive cell surface proteius obtained from AT&= or ATCr,e were mixed with 50 ~1 of carrier KLH or FYG and 50 ~1 rabbit of undiluted anti KLH or anti FyG antiserum. The amount of carrier was determined previously using trace amounts of 125I (iodide) labelled antigen, various dilutions of carrier and a constant amount of antiserum. In these experiments the addition of 1 pg of FyG carrier and 10 pg of KLH to the respective antisera resulted in precipitations of 80-90s of the antigen. These precipitating systems served as indicators of binding specificity for antigen when incubated with surface protein from cells activated to the homologous antigen and as indifferent precipitate specificity controls in the presence of surface protein of cells activated to the heterologous antigen.
CELL
INTERACTIONS
IV2
vitt-0
BY
T
CELL
IgM
5
200-
RELATIVE
MOBILITY
FIG. 2. Analysis by polyacrylamide gel electrophoresis in acid urea of polypeptide chains of specifically coprecipitated surface immunoglobulin from T-cells activated to KLH. 0 0 surface immunoglobulin reduced and alkylated in the presence of 9 M urea. 6% gel. B, y and L refer to the positions of reference immunoglobulin polypeptide chains ; P, p chain; y, Y chain; L, light chain. The counts given are specific counts. The coprecipitation controls using normal rabbit serum were subjected to polyacrylamide gel electrophoresis under identical conditions. These nonspecific values were subtracted at each point from those of the specifically precipitated material.
RESULTS Characterization
of Activated
T Cell Surface
Immunoglobulin
To recover surface immunoglobulin molecules under gentle conditions, such that binding studies could be performed, proteins released by membrane turnover from the cell surface were isolated (25). KLH-activated T cells were iodinated with lzsI (iodide) to high specific activity using the lactoperoxidase technique (12). The radioiodinated cells were incubated for 3 hr in tissue culture. After centrifugation the cells were discarded and the supernatants were retained. Immunoglobulin was detected in the supernatants by specific coprecipitation. Approximately 6% of radioactively labeled non dialyzable surface proteins present in the supernatant were specifically coprecipitated. Complexes of specifically precipitated radioiodinated cell surface proteins and antiglobulins were dissolved in 9 M urea-lo% acetic acid. The solubilized immunoglobulin was analysed by PAGE under conditions in which intact IgG immunoglobulin exhibited a relative mobility of 0.33. As Fig. 1 shows, the intact cell surface immunoglobulin migrated as a single component which possessed an RF slightly lower than that of the specifically coprecipitated mouse IgG standard. This result was consistent with our previous findings by gel filtration that surface immunoglobulin of thymus cells was monomeric TgM with a molecular weight of approximately 180,000 (15, 16). We studied the polypeptide chain structure of surface immunoglobulin from KLH-activated T cells by dissolving the specific coprecipitates in 9 M urea then reducing and alkylating to cleave interchain disulfide bonds. The polypeptide chains were analysed by PAGE performed under conditions which resolved light chains, chains and p chains (6). Two poly-
FELDMANN,
6
CONE, AND TABLE
MARCHALONIS 1
COPRECIPITATION OF ACTIVATED T Cer.r. SUKFACE BY ANTIBODY TO ACTIVATING; ANTIGENY
Cell surface proteins f rom : KLH-activated
T cells
1. 2. 3. 4.
Coprecipitation conditions
Counts,/ minute _____
KLH + anti-KLH KLH + NRS F,G + anti-F,G MrG + anti-MIg
936 130 90 3200
f f f i
Sign&axe
224 30 25 150
KLH-activated T cells, cell surface proteins coprecipitated with anti-Ig
5. KLH 6. KLH
+ anti-KLH + NRS
43 * 15 20 f 10
KLH-activated T cells, cell surface proteins coprecipitated with NRS
7. KLH 8. KLH
+ anti-KLH + NRS
870 f 90 55 zk 20
F,G-activated
T cells
9. 10. 11. 12.
F,G + anti-F& F,G + NRS KLH + anti-KLH MIG + anti-MIg
337 55 94 1100
PKUTEIN
z!z 12 i 5 4~ 38 A 200
1 and 2 or 3 P < 0.01 2 and 3 P > 0.05
1 and 5 P < 0.001 5 and 6 F > 0.05
7 and 8 P < 0.01
9 and 10 or 11 P < 0.01 10 and 11 P > 0.05
a Cell surface proteins were prepared by incubation of 2-3 X 10 7 ‘251-labelled activated T lymphocytes in 2 ml tissue culture medium for 3 hr at 37°C. The cells were centrifuged and the supernatants saved for coprecipitation analysis. Data represent the arithmetic mean f SE of 3 or 4 replicate samples. Background radioactivity has been subtracted from all values. P values computed by Student’s t-test and modified for small samples (37). NRS-normal rabbit serum; antiMIg-rabbit anti-mouse immunoglobulin antiserum.
peptide components were demonstrated. One resembled the light chains of standard immunoglobulin in gel penetration the other was similar to the p heavy chain in mobility. No components which migrated in the region of y chains was observed (Fig. 2). This polypeptide chain structure is similar to that reported for thymus cells activated against histocompatibility antigens ( 18). Specificity
of Activated
T Cell Surface
Immunoglobulin
fog the Activating
Antigen
The antigen binding specificity of cell surface immuuoglobulin from the activated cells was estimated by coprecipitation with the activating antigen and rabbit antisera to the antigen. As shown in Table 1, I*‘1 labeled cell surface proteins were coprecipitated with an antigen-antibody system that contained the antigen used to activate the cells and antibody to that antigen. Thus, cell surface proteins from KLH activated cells were precipitated with a KLH anti KLH system and surface proteins from FyG activated cells were precipitated with a FyG anti FyG system. Controls showed that mouse imusing lz51 ( iodide) labeled purified mouse immunoglobulin munoglobulin is not precipitated by either KL,H-anti KLH or anti F,C-F,G. If cell surface immunoglobulin was removed prior to the antigen binding assay, lz51 (iodide) cell surface proteins were not precipitated.
CELL
The Bind&g
INTERACTIONS
I?2
Cafiacity of the Collaborative
vit?‘O
Factor
BY
T
CELL
7
IgM
to Lymphoid
Cells Classes
Lymphocytes as well as macrophages have receptors for immunoglobulin (e.g., 28). For example, Basten has demonstrated that antigen-antibody complexes bind firmly to B lymphocytes (29). Although it was demonstrated previously that peritoneal exudate cells and purified macrophages bound the collaborative factor (lo), the possibility that other cells could also bind this factor was not excluded. Experiments were thus performed to compare the capacity of macrophages and T or B lymphocytes to bind the collaborative factor derived from ATC in an immunogenic manner. AT&n were cultured in the upper compartment of double chamber flasks, together with the antigen DNP KLH, and either peritoneal exudate cells, CBA thymocytes as a source of T cells, column purified lymphocytes, or nu/nu spleen cells as a source of B cells were incubated in the lower compartment for 24 hours. After this time the cells in the lower compartment were harvested, washed, and lo5 viable cells were added to cultures of DNP Fla primed spleen cells. The antibody responses to DNP and DRC were assayed 4 days later (Table 2). In this experimental system, DNP Fla primed spleen cells will make anti DNP antibody only if they are cultured with T cells activated to the carrier antigen, in this case, KLH (7). Significant responses were obtained only when peritoneal exudate cells which had been initially cultured with ATC KC~ were transferred to DNP Fla primed spleen cells. Those results suggested that the collaborative factor only bound effectively to macrophages. Binding
of Activated
T Cell Surface
Inzwaunoglobulin
to Macrophages
The experiments described above suggested that immunoglobulin released by T cells is cytophilic for macrophages in the presence of antigen. To test whether the isolated molecules would bind to macrophages, lzzI (iodide) labeled cell surface proteins were incubated with peritoneal macrophages, thymus lymphocytes or spleen cells from nu/nu mice. As shown in Table 3 cell surface proteins from activated T cells bound significantly to macrophages but did not bind appreciably to thymus lymphocytes or nu/nu spleen cells. If immunoglobulin was removed by coprecipita-
TABLE
2
BINDING OF THE COLLABOKATIVE FACTOR TO LYMPHOID CELL CLASSES~ Cells cultured Upper
Antibody response (AFC/culture f SE)
Lower DNP
ATCma
Peritoneal exudate Nu/nu spleen cells CBA thymus Lymphocytes
720 + 160 40 f 20 0 0
DRC 2640 2070 1690 2870
+ f zk i
120 180 360 395
D 2 X lo6 ATCKLH were cultured in the upper chamber, with 2 X lo6 other cells in the lower chamber and 1 fig/ml DNP KLH for 24 hours. Lymphocytes were obtained by column filtration (23). lo6 of the cells cultured in the lower chamber were cultured with 1.5 X 107 DNP Fla primed spleen cells for 4 days. Values represent arithmetic mean f the standard error of the mean of 4 cultures.
8
FEZDMANN,
CONE,
AND
MARCHALONIS
tion prior to the binding assay little or no radioactivity was bound to the cells. After correction for losses in washing, at least 705% of the radioactivity precipitated as immunoglobulin was specifically absorbed by macrophages. To determine whether T cell antigen receptors, bound to macrophages in the absence of antigen, could subsequently mediate cell cooperation, AT&,= were cultured for 2 days in the upper chamber of double flasks either with DNP KLH or DNP FyG, and macrophages cultured in the lower chamber. These macrophages were harvested, washed and cultured with DNP Fla primed spleen cells in the presence of antigen, DNP KLH (Table 4). An anti-DNP response occurred with the addition of DNP KLH if ATCRLH were used in vitro, hut not if ATCFVc were cultured. These results indicated that the macrophagcs did bind a T cell product, which enabled them to subsequently bind specific antigen and immunize B cells. However, these macrophages did not induce as high antibody responses as those which bound receptor-antigen complexes. Cooperation
After
Initial
Binding
of Antigen
to T Cells
Cell interaction of T and B lymphocytes across the nucleopore membrane is specific for antigenic determinants which are linked to those recognised by T cells (7, 10). Since the T cell specificity of cooperation was the same as that of T cell activation, the T cell component should have the same specificity as the T cell receptors. To determine whether T cell receptors for antigen could participate in the process of cell cooperation, ATC RLH were first incubated for 1 hr at 4°C with 1 pg/ml DNP KLH, washed thoroughly and then cultured in double chambers with peritoneal exudate cells in the lower compartment, in the absence of further antigen (Table 5). The cooperative capacity of the prcincubated cells was the same as that when antigen was prcscnt for the entire period of initial culture. These results suggested that at least part of the mediator of cell cooperation was by T
TABLE
3
BINDING OF lzSI (iodide) KLH ACTIVATED T CELL PROTEINS TO VARIOUS CELL TYPEF Cell surface protein treatment
lzsI (iodide) Macrophages
Thymus
SURFACE
counts bound to: B cells~
cells
None
1. 1445 f
263
2.
55 f
26
3.
Coprecipitated with antiimmunoglobuliu
4. 33.8 f
33.3
5. 81.6 f
5.1
6.
Coprecipitated NRS
7. 1448 f
307
8.
123 i
53
9. 69.3 f
with
Significance
94 + 15 0
23
1 P 4 I’ 4 7
and 2, 3 or 4 < 0.001 and 7 < 0.001 and 5 P > 0.05 and 8 or 9 P < 0.001
a 100 ~1 of lzsI (iodide)-labeled cell surface proteins were incubated with 2 X lo6 macrophages, thymus cells or B cells in 100 ~1 PBS for 2 hours at 4OC. The cells were washed 3-6 times and radioactivity in the pellet determined. Data are represented as counts per minute. Results represent the arithmetic mean ZIZSE of values obtained in 3 experiments with 3-4 replicates per group in each experiment. Background radioactivity has been subtracted from all values. b B cells = 2 X 106 nu/nu spleen cells.
cm.L
INTERACTIONS
In Vitro TABLE
BY
T
CELL
9
IgM
4
CELL COOPERATION WITH SEQUENTIAL BINDING OF THE T CELL COMPONENT, THEN ANTIGEN TO MACROPHAGES Anti-DNP response AFC/culture
Cells cultured Lower
Upper DNP DNP DNP
ATCKLH ATCKLH AT&G
KLH F,G KI,H
Macrophages Macrophages Macrophages
780 f 105 340 f 956 0
a 2 X 106ATCmn or ATCs-x: were cultured in the upper chamber with either 1 rg/ml or DNP F,G for 2 days with peritoneal exudate cells as a source of macrophages chamber. IO5 peritoneal exudate cells were cultured with 1.5 X 10’ DNP Fla primed for 3 days in the presence of 1 pg/ml DNP KLH. Values represent the arithmetic mean error of 4 cultures. * P < 0.05 compared with group initially cultured with DNP KLH.
cell receptor-antigen, already complexed, absorbed by the peritoneal exudate cells.
DNP KLH in the lower spleen cells f standard
released by the T cells and subsequently
DISCUSSION The experiments presented here were designed to ascertain the chemical nature of the factor released by T cells which participates in specific cooperation with B cells. The cell surfaces of activated T cells used in in vitro cooperative systems (6, 7) were labeled with 1Z51-iodide (12). These were shown to possess surface immunoglobulin with light chains, and heavy chains which resembled p chains when analyzed by polyacrylamide gel electrophoresis in acid urea. The gel electrophoretic mobility of the intact immunoglobulin was consistent with a molecular weight of approximately 180,000 for the protein. These results corresponded well to previous results obtained for surface immunoglobulin of T cells activated to histocompatibility antigens (18). Furthermore, the presence of light chains and TABLE
5
COOPERATION ACROSS A MEMBRANE AFTER INITIAL ANTIGEN TO ATCa Antigen
DNP
KLH
DNP F,G
BINDING UF
Duration
Anti-DNP Response (AFC/culture ZJZSE)
2 days 1 hr 2 days 1 hr
580 f 140 490 f 100 0 0
(i 2 X lo6 ATCKL~ were incubated with 1 rig/ml DNP KLH or DNP,FG for 1 hour at 4”C, washed. To half of each group of cells antigen was readded. The ATCmn were then cultured in double chamber flasks with peritoneal exudate cells in the lower compartment. IO5 macrophages from each group were then cultured for 3 days with DNP Fla primed spleen cells. Values are the arithmetic mean of 4 cultures & the standard error of the mean of 4 cultures, Similar results were obtained in 2 other experiments.
10
FELDMANN,
CONE,
AND
MARCHALONIS
p chains confirmed prior results demonstrating that the factor mediating T cell and B cell interaction in the in vitro systems was specifically inhibited by treatment with rabbit antisers to murine K-chains and p-chains (10). The binding specificity of the isolated surface immunoglobulin for the activating antigen was established by coprecipitation assay (Table 1) . The capacity of the surface IgM of ATC to bind various cell classes was investigated (Table 3) and found to correlate precisely with the binding of the collaborative factor (Table 2). These observations suggested that the T cell component of the collaborative factor resembled the specific receptor of ATC. Evidence to support this concept can be marshalled as follows: (a) AT&H binds sufficient DNP-KLH in 60 minutes at 4°C to collaborate efficiently with B cells, with adherent cells as an intermediate (Table 5) ; (b) metabolic inhibitors abrogate both collaboration by ATC (8) , and the release of surface immunoglobulin (25) ; (c) macrophages bind both T cell surface IgM (Table 3), and the T cell component which, in the presence of antigen, induces antibody responses (Table 2, Ref. 10). The combination of biochemical and tissue culture data presented above is consistent with the hypothesis of Bretscher and Cohn (30) that specific T/B lymphocyte interaction involves “carrier or associative antibody.” We provide evidence that this carrier antibody has a MW of approximately 180,000 and contains p and light chains. The molecule can be described as a monomeric IgM immunoglobulin. However, there is as yet no data on the cell synthesizing the collaborative factor (or T cell monomeric IgM) . The discovery of carrier antibody as one of the T cell components of cell interaction has posed some intriguing questions. Firstly, it is known that B cells possess on their surfaces monomeric IgM immunoglobulin which is probably receptor for antigen (14, 16, 17). Moreover, these molecules turn over in a relatively rapid fashion, compared to T cell IgM immunoglobulin (18, 31, 32). However because B cells have not yet been shown to act as helper cells (33, 34) it is likely that B cell monomeric IgM does not function in cell cooperation. Since monomeric IgM must bind to adherent cells (lo), perhaps macrophages or dendritic cells (35) in order to mediate cell interaction, the inability of B cells to collaborate may be explained by the different binding characteristics of the B cell receptor. Recent experiments indicate that B cell monomeric IgM does not bind to macrophages (38). Secondly, the dual function of the T cell receptor may prove to be of key biological interest. This monomeric IgM not only acts as a receptor for antigen, but also acts as a transmitter of immunological information, extending the range of action of T cells in cooperation. This amplification property of released T cell receptor obviates a major weakness of models of interaction between T cells and B cells which require contact between two rare cell types (36). These two distinct functions of a single receptor molecule are thus far unique in biological systems. ACKNOWLEDGMENTS The technical assistance of Mrs. J. Thompson, Misses B. Pike, J. Jackson, P. Smith J. Gamble is gratefully acknowledged.
and
CELL
INTERACTIONS
I?2
YitfV
BY
T CELL
IgM
11
REFERENCES 1. 2. 3. 4. 5.
Miller, J. F. A. P., and Mitchell, G. F., Trunsplallt. Ziev. 1, 3, 1969. Davies, A. J. S., Transplant. Rev. 1, 43, 1969. Claman, H. N., and Chaperon, E. A., Transplant. Rev. 1, 92, 1969. Mitchison, N. A., Eur. J. Zmmunol. 1, 10, 1971. Cheers, C., Breitner, J. C. S., Little, M., and Miller, J. F. A. P., Nature New Biology 232, 248, 1971. 6. Feldmann, M., and Basten, A., Nature New Biology 237, 13, 1972. 7. Feldmann, M., and Basten, A., J. Exp. Med. 156, 49, 1972. 8. Feldmann, M., and Basten, A., Eur. J. Imrnwzol. 2, 213, 1972. 9. Feldmann, M., J. Exp. Med. 135, 1049, 1972, 10. Feldmann, M., /. Erp. Med. 136, 737, 1972. 11. Marchalonis, J. J,, Biochem. J. 113, 299, 1969. 12. Marchalonis, J. J., Cone, R. E., and Sante, V., Biochem. J. 124, 921, 1971. 13. Phillips, D. R., and Morrison, M., Biochemistry 10, 1766, 1971. 14. Baur, S., Vitetta, E. S., Sher, C. J., Schenkein, I., and Uhr, J. W., J. Zmmunol. 106, 1133, 1971. 15. Marchalonis, J. J., Atwell, J. L., and Cone, R. E., Nature (Londor~) 235, 240, 1972. 16. Marchalonis, J. J., Cone, R. E., and Atwell, J. L., Proc. Nat. Acnd. Sci. U.S.A. (in press), 1972. 17. Vitetta, E. S., Baur, S., and Uhr, J. W., J. Exp. Med. 134, 242, 1971. 18. Marchalonis, J. J., Cone, R. E., Atwell, J. L., and Rolley, R. T., Ztz “The Biochemistry of Gone Expression in Higher Organisms” (in press), 1972. 19. Feldmann, M., Wagner, H., Basten, A., and Holmes, M., Aust. J. Exp. Biol. Med. 56, 651, 1972. 20. Miller, J. F. A. P., and Warner, H. L., Znt. Arch. All. 40, 59, 1971. 21. Feldmann, M., J. Exp. Med. 135, 735, 1972. 22. Cunningham, A. J., and Szenberg, A., Immunology 14, 599, 1968. 23. Shortman, K., Williams, R., Jackson, R., Russell, P., Byrt, P., and Diener, R., /. Cell. Biology
48, 566, 1971.
24. Shortman, K., Williams, N., and Adams, P., J. Zmmunol. Methods (in press), 1972. 25. Cone, R. E., Marchalonis, J. J., and Rolley, R. T., J. Exp. Med. 134, 1373, 1971. 26. Edelman, G. M., and Marchalonis, J. J., In “Methods in Immunology and Immunochemistry (C. A. Williams and H. W. Chase, Eds.), Vol. 1, p. 405. Academic Press, New York, 1967. 27. Parish, C. R., and Marchalonis, J. J., Asal. Biochem. 34, 436, 1970. 28. Philips-Quagliata, Levine, B. B., Quagliata, F., and Uhr, J. W., J. Exp. Med. 133, 589, 1971. 29. Baston, A., Miller, J. ‘F. A. P., Sprent, J., and Pye, J., J. Exp. Med. 135, 610, 1972. 30. Brestcher, P. A., and Cohn, M., Nature (London) 220, 444, 1968. 31. Wilson, J. D., Nossal, G. J. V., and Lewis, H., Eur. 1. Zmnzunol. 2, 225, 1972. 32. Lerner, R. A., McConahey, P. J., Jansen, I., and Dixon, F. J., 1. Exp. Med. 135, 136, 1972. 33. Feldmann, M., Eur. J. Zntmm~tol. 2, 130, 1972. 34. Miller, J. F. A. P., Sprent, J., Basten, A., Warner, N. L., Sreitzer, J. C. S., Rowland, G., Hamilton, J., Silver, H., and Martin, W. J., J. Exp. Med. 134, 1266, 1971. 3.5. Guttman, G., and Weissman, I. L., Z~nmunologg 23, 465, 1972. 36. Mitchison, N. A., Taylor, R., and Rajewsky, K., I?$ “Developmental Aspects of Antibody Formation and Structure” (J. Sterzl, Ed.), p. 517. Publ. House. Czech. Acad. Sci., Prague, 1970. 37. Bailey, N. T. J., “Statistical Methods in Biology” p. 43. English Univ. Press Ltd. London, 1959. 38. Cone, R. E., ‘Feldmann, M., Marchalonis, J. J., and Nossal, G. J. V. Zmmunology (in press), 1973.
IMMUNOLOGY
Cell
9,
l-11
(1973)
Interactions
in the Immune
VI. Mediation
by T Cell Surface
P\/IAKc
Walter
J.
MARCIIALONIS~
Iustitute Hospital,
of Medical Melbourne,
JOHN
and E&a Hall Royal Melbourne
Monomeric
Received
IgM1
ROBERT E. CONE,~
FIXDMANN,” AND
Response in Vitro
August
Research, Post 3050, Australia
Ofice,
11, 1972
The nature of the specific mediator of T/B lymphocyte cooperation was investigated by using biochemical and in vitro culture methods in parallel. By surface radioiodination of activated thymus cells it was found that the T cell component of cell interaction was an immunoglobulin which possessed a mobility on polyacrylamide gel electrophoresis consistent with a molecular weight of approximately 180,000. This mobility of molecule comprised of two polypeptide chains, with the electrophoretic light and p chains. This monomeric IgM molecule had specificity for the activating antigen, and like the mediator of cell interaction, bound cytophilically to macrophages, but not to T or B lymphocytes. These studies extend previous work in which the mediator of cell cooperation was found, by the use of antisera to contain antigen, and K and p chain determinants. The binding of the T cell surface IgM to macrophages supports the concept that the final step of cell cooperation, the immunization of B cells occurs at the surface of macrophages, occurs via a lattice of antigenic determinants created by cytophilic T cell carrier antibody.
INTRODUCTION Collaboration between thymus derived (T) and non thymus derived (B) lymphocytes is required for optimal antibody responses to many antigens, such as heterologous erythrocytes or hapten protein conjugates (1-5). The mechanisms by which T cells facilitate antibody production by B cells has recently been investigated in vitro. It was found that direct contact of T and B lymphocytes was not essential (6, 7)) but that active T cell metabolism was required (8). These results suggested that lymphoid cell collaboration was mediated by a released subcellular T cell component. Furthermore, it was found that adherent cells presumably macrophages were required for the production of antibody in vitro to thymus dependent antigens, even to those of small size, whereas adherent cells were not essential for antibody 1 This work was supported by grants from the National Health and Medical Research Council, Canberra, Australia, the Australian Research Grants Committee, the National Institute of Allergy and Infectious Disease (AI-o-3958) and the American Heart Association (721050). This is publicatoin No. 1720 from the Walter and Eliza Hall Institute. a Present address : Tumour Immunology Unit, Department of Zoology, University College London, England. * Postdoctoral fellow of the Damon Runyon Memorial Fund for Cancer Research. l Address requests for reprints to: J. J. Marchalonis, Walter and Eliza Hall Institute of Medical Research, Post Office, Royal Melbourne Hospital, Melbourne, 3050, Australia.
Copyright All rights
0 1973 by Academic Press, of reproduction in any form
Inc. reserved.
2
FELDMANN,
CONE,
AND
MARCHALONIS
production to thymus independent antigens (9). This observation suggested that macrophages or macrophage-like cells are involved in lymphocyte cooperation. This was confirmed by culturing these cells, separated by a cell impermeable nucleopore membrane, from antigen activated thymus cells (ATC) and antigen. In this system it was shown that the macrophages had acquired the capacity to induce antibody formation in B cell populations selectively to the antigen used to activate the T cells (10). Because the macrophages only acquired immunogenic capacity in the presence of specific ATC, it appeared that the macrophages were influenced by a T cell subcellular component (10). Trypsinization of the macrophages which had been cultured with ATC and antigen demonstrated that the immunogenic moiety was present on the cell surface. Serologic studies using anti-antigen and antiimmunoglobulin heavy and light chain antisera indicated that the specific mediator of cell collaboration was a complex of T ccl1 IgM and antigen (10). Lactoperoxidase-catalysed radioiodination (11) of cell surface proteins (12-14) has enabled the isolation of cell surface immunoglobulin from both T (15, 16) and B (14, 16, 17) lymphocytes. T cell surface immunoglobulin is a monomeric IgM molecule (15, 16, 18) which, when obtained from activated cells possesses binding specificity for the activating antigen (18). Thus it seemed probable that the T cell IgM involved in cell collaboration was similar to the T cell receptor for antigen. In the present communication, parallel immunological and biochemical evidence is presented which identifies the T cell component of the collaborative factor as a monomeric IgM molecule. MATERIALS
AND
METHODS
Aniwds CBA/H/Wehi mice were used for most experiments. Congenitally athymic (nu/nu) mice provided a source of lymphoid cells devoid of T lymphocytes Activation
of Thyrnus
nude (19).
Cells
Lethally irradiated (800 rad) mice were injected with syngeneic thymocytes and antigen, as described elsewhere (8). Six to seven days later their spleens served as a source of activated thymus cells (ATC) . Antigens Keyhole limpet hemocyanin (KLH) was obtained from Calbiochem, Sydney, Australia. Fowl gammaglobulin (FyG) was prepared as described by Miller and Warner (20). The dinitrophenyl (DNP) determinant was conjugated to KLH, FyG, and flagella of Salmonella adelaide (Fla) as described elsewhere (21) . Inwnunization CBA mice were injected intraperitoneally were used in culture l-3 months later. Tissue
with
25 pg DNP
Fla. Their
spleens
Culture
Mouse spleen cells were cultured in a modified Marbrook-Diener For routine generation of responses, single compartment culture
culture system. flasks were used.
CELL
INTERACTIONS
In
Vitro
BY
T
CELL
IgM
3
Cultures with 2 compartments were performed as described elsewhere (7). The two compartments were separated by a cell impermeable nuclepore membrane of 0.2 0 pore size (Nuclepore, General Electric Co., U.S.A.). ATC were placed in the upper compartment and various cell populations in the lower chamber. Media and sera used for culture were as in previous communications (7) _ Enumeration
of Antibody
Forming
Cells (AFC)
Cells forming antibody were enumerated by the Cunningham and Szenberg fication (22) of the hemolytic plaque assay, as described elsewhere (21). Peritoneal
Exudate
Six to eight months proteose peptone broth 4-5 days later. Ma.crophage
modi-
old CBA mice were injected (Difco, Michigan). Peritoneal
intraperitoneally with 1 ml exudate cells were obtained
Depletion
The active adherence column technique of Shortman et al. (23) was used. Basically, spleen cells suspended in 50% mouse serum are passed rapidly through a column of large siliconized glass beads at 37°C. The effluent is extensively depleted of phagocytes (23). Antisera Rabbit antiserum to KLH was prepared by immunizing a rabbit twice with 1 mg KLH emulsified in Freund’s complete adjuvant. Rabbit anti fowl gamma globulin antiserum was generously provided by Dr. B. T. Rouse. This antiserum bound to IgM and to the 7s immunoglobulin of the chicken. Preparation
of Cell Surface
Imwtunoglobulin
Activated T cells were sedimented through bovine serumatin (BSA) gradients (24) to remove dead cells and debris. The cells were then washed 5 times in phosphate-buffered saline, pH 7.2 and iodinated to high specific activity using lactoperoxidase catalyzed radioiodination (12). Briefly, lo7 cells were suspended in 50 ~1 lactoperoxidase (25 pg) and 200 &i W (iodide) and 10 ~1 of 0.03% H202 were added. The reaction was initiated by vigourous shaking and the cells washed twice with PBS. Fifty to 100 million cells were iodinated in aliquots of 1 x 10’ cells. After iodination the cells were incubated Lmder short-term cell culture conditions for 3 hr, centrifuged and the supernatants retained as described previously (15, 16). Under these conditions, cell surface proteins, including immunoglobulin are released into the cell culture medium (16, 2.5). Radioactive cell surface immunoglobulin was isolated from the supernatants by specific coprecipitation using rabbit anti mouse immunoglobulin antiserum and purified mouse G immunoglobulin as carrier (15, 16). The rabbit antiserum reacted with light chains and p heavy chains. Radioactivity was determined in a Packard Autogamma scintillation counter with a deep well Nal crystal detector.
FELDMANN,
4
CONE,
AND
MARCHALONIS
FIG. 1. Analysis by polyacrylamide gel electrophoresis in acid urea of specifically coprecipitated surface immunoglobulin from T cells activated to KLH. Intact immunoglobulins resolved on 0, surface immunoglobulin from activated T cells; O- - - -0, specifically 4% gel. 0 coprecipitated mouse rG immunoglobulin. Under those conditions 19s IgM does not enter the gel.
Resolution (PAGE)
of Cell Surface
Iwwnunoglobulin
by Polyacrylanzide
Gel Electrophoresis
The molecular size of intact cell surface immunoglobulin was determined by dissolving coprecipitated immunoglobulin in 9 M urea, 10% acetic acid. Coprecipitated immunoglobulins were also dissolved in 9-M urea and reduced and alkylated to cleave interchain disulfide bonds (26). Intact and reduced and alkylated samples and immunoglobulin markers were resolved by disc electrophoresis in acid urea (27). Cell and Antigen
Binding
Assays
z&h
Cell Surface
Proteins
The ability of ATC cell surface proteins to bind to various cell types was determined by adding 100 ~1 of radioactive cell surface proteins to 2 X lo6 CBA peritoneal macrophages, CBA thymus cells or nu/nu spleen cells in 100 ~1 phosphate buffered saline (PBS, pH 7.2) containing 10% FCS. The cells were incubated for 1 hr at 4°C and washed 46 times with PBS containing 10% FCS. Radioactivity was monitored in the pellet after each wash. The antigen binding specificity of ATC cell surface proteins was determined by coprecipitation with the activating antigen and a rabbit antiserum to the antigen. Briefly, 100 ~1 of radioactive cell surface proteius obtained from AT&= or ATCr,e were mixed with 50 ~1 of carrier KLH or FYG and 50 ~1 rabbit of undiluted anti KLH or anti FyG antiserum. The amount of carrier was determined previously using trace amounts of 125I (iodide) labelled antigen, various dilutions of carrier and a constant amount of antiserum. In these experiments the addition of 1 pg of FyG carrier and 10 pg of KLH to the respective antisera resulted in precipitations of 80-90s of the antigen. These precipitating systems served as indicators of binding specificity for antigen when incubated with surface protein from cells activated to the homologous antigen and as indifferent precipitate specificity controls in the presence of surface protein of cells activated to the heterologous antigen.
CELL
INTERACTIONS
IV2
vitt-0
BY
T
CELL
IgM
5
200-
RELATIVE
MOBILITY
FIG. 2. Analysis by polyacrylamide gel electrophoresis in acid urea of polypeptide chains of specifically coprecipitated surface immunoglobulin from T-cells activated to KLH. 0 0 surface immunoglobulin reduced and alkylated in the presence of 9 M urea. 6% gel. B, y and L refer to the positions of reference immunoglobulin polypeptide chains ; P, p chain; y, Y chain; L, light chain. The counts given are specific counts. The coprecipitation controls using normal rabbit serum were subjected to polyacrylamide gel electrophoresis under identical conditions. These nonspecific values were subtracted at each point from those of the specifically precipitated material.
RESULTS Characterization
of Activated
T Cell Surface
Immunoglobulin
To recover surface immunoglobulin molecules under gentle conditions, such that binding studies could be performed, proteins released by membrane turnover from the cell surface were isolated (25). KLH-activated T cells were iodinated with lzsI (iodide) to high specific activity using the lactoperoxidase technique (12). The radioiodinated cells were incubated for 3 hr in tissue culture. After centrifugation the cells were discarded and the supernatants were retained. Immunoglobulin was detected in the supernatants by specific coprecipitation. Approximately 6% of radioactively labeled non dialyzable surface proteins present in the supernatant were specifically coprecipitated. Complexes of specifically precipitated radioiodinated cell surface proteins and antiglobulins were dissolved in 9 M urea-lo% acetic acid. The solubilized immunoglobulin was analysed by PAGE under conditions in which intact IgG immunoglobulin exhibited a relative mobility of 0.33. As Fig. 1 shows, the intact cell surface immunoglobulin migrated as a single component which possessed an RF slightly lower than that of the specifically coprecipitated mouse IgG standard. This result was consistent with our previous findings by gel filtration that surface immunoglobulin of thymus cells was monomeric TgM with a molecular weight of approximately 180,000 (15, 16). We studied the polypeptide chain structure of surface immunoglobulin from KLH-activated T cells by dissolving the specific coprecipitates in 9 M urea then reducing and alkylating to cleave interchain disulfide bonds. The polypeptide chains were analysed by PAGE performed under conditions which resolved light chains, chains and p chains (6). Two poly-
FELDMANN,
6
CONE, AND TABLE
MARCHALONIS 1
COPRECIPITATION OF ACTIVATED T Cer.r. SUKFACE BY ANTIBODY TO ACTIVATING; ANTIGENY
Cell surface proteins f rom : KLH-activated
T cells
1. 2. 3. 4.
Coprecipitation conditions
Counts,/ minute _____
KLH + anti-KLH KLH + NRS F,G + anti-F,G MrG + anti-MIg
936 130 90 3200
f f f i
Sign&axe
224 30 25 150
KLH-activated T cells, cell surface proteins coprecipitated with anti-Ig
5. KLH 6. KLH
+ anti-KLH + NRS
43 * 15 20 f 10
KLH-activated T cells, cell surface proteins coprecipitated with NRS
7. KLH 8. KLH
+ anti-KLH + NRS
870 f 90 55 zk 20
F,G-activated
T cells
9. 10. 11. 12.
F,G + anti-F& F,G + NRS KLH + anti-KLH MIG + anti-MIg
337 55 94 1100
PKUTEIN
z!z 12 i 5 4~ 38 A 200
1 and 2 or 3 P < 0.01 2 and 3 P > 0.05
1 and 5 P < 0.001 5 and 6 F > 0.05
7 and 8 P < 0.01
9 and 10 or 11 P < 0.01 10 and 11 P > 0.05
a Cell surface proteins were prepared by incubation of 2-3 X 10 7 ‘251-labelled activated T lymphocytes in 2 ml tissue culture medium for 3 hr at 37°C. The cells were centrifuged and the supernatants saved for coprecipitation analysis. Data represent the arithmetic mean f SE of 3 or 4 replicate samples. Background radioactivity has been subtracted from all values. P values computed by Student’s t-test and modified for small samples (37). NRS-normal rabbit serum; antiMIg-rabbit anti-mouse immunoglobulin antiserum.
peptide components were demonstrated. One resembled the light chains of standard immunoglobulin in gel penetration the other was similar to the p heavy chain in mobility. No components which migrated in the region of y chains was observed (Fig. 2). This polypeptide chain structure is similar to that reported for thymus cells activated against histocompatibility antigens ( 18). Specificity
of Activated
T Cell Surface
Immunoglobulin
fog the Activating
Antigen
The antigen binding specificity of cell surface immuuoglobulin from the activated cells was estimated by coprecipitation with the activating antigen and rabbit antisera to the antigen. As shown in Table 1, I*‘1 labeled cell surface proteins were coprecipitated with an antigen-antibody system that contained the antigen used to activate the cells and antibody to that antigen. Thus, cell surface proteins from KLH activated cells were precipitated with a KLH anti KLH system and surface proteins from FyG activated cells were precipitated with a FyG anti FyG system. Controls showed that mouse imusing lz51 ( iodide) labeled purified mouse immunoglobulin munoglobulin is not precipitated by either KL,H-anti KLH or anti F,C-F,G. If cell surface immunoglobulin was removed prior to the antigen binding assay, lz51 (iodide) cell surface proteins were not precipitated.
CELL
The Bind&g
INTERACTIONS
I?2
Cafiacity of the Collaborative
vit?‘O
Factor
BY
T
CELL
7
IgM
to Lymphoid
Cells Classes
Lymphocytes as well as macrophages have receptors for immunoglobulin (e.g., 28). For example, Basten has demonstrated that antigen-antibody complexes bind firmly to B lymphocytes (29). Although it was demonstrated previously that peritoneal exudate cells and purified macrophages bound the collaborative factor (lo), the possibility that other cells could also bind this factor was not excluded. Experiments were thus performed to compare the capacity of macrophages and T or B lymphocytes to bind the collaborative factor derived from ATC in an immunogenic manner. AT&n were cultured in the upper compartment of double chamber flasks, together with the antigen DNP KLH, and either peritoneal exudate cells, CBA thymocytes as a source of T cells, column purified lymphocytes, or nu/nu spleen cells as a source of B cells were incubated in the lower compartment for 24 hours. After this time the cells in the lower compartment were harvested, washed, and lo5 viable cells were added to cultures of DNP Fla primed spleen cells. The antibody responses to DNP and DRC were assayed 4 days later (Table 2). In this experimental system, DNP Fla primed spleen cells will make anti DNP antibody only if they are cultured with T cells activated to the carrier antigen, in this case, KLH (7). Significant responses were obtained only when peritoneal exudate cells which had been initially cultured with ATC KC~ were transferred to DNP Fla primed spleen cells. Those results suggested that the collaborative factor only bound effectively to macrophages. Binding
of Activated
T Cell Surface
Inzwaunoglobulin
to Macrophages
The experiments described above suggested that immunoglobulin released by T cells is cytophilic for macrophages in the presence of antigen. To test whether the isolated molecules would bind to macrophages, lzzI (iodide) labeled cell surface proteins were incubated with peritoneal macrophages, thymus lymphocytes or spleen cells from nu/nu mice. As shown in Table 3 cell surface proteins from activated T cells bound significantly to macrophages but did not bind appreciably to thymus lymphocytes or nu/nu spleen cells. If immunoglobulin was removed by coprecipita-
TABLE
2
BINDING OF THE COLLABOKATIVE FACTOR TO LYMPHOID CELL CLASSES~ Cells cultured Upper
Antibody response (AFC/culture f SE)
Lower DNP
ATCma
Peritoneal exudate Nu/nu spleen cells CBA thymus Lymphocytes
720 + 160 40 f 20 0 0
DRC 2640 2070 1690 2870
+ f zk i
120 180 360 395
D 2 X lo6 ATCKLH were cultured in the upper chamber, with 2 X lo6 other cells in the lower chamber and 1 fig/ml DNP KLH for 24 hours. Lymphocytes were obtained by column filtration (23). lo6 of the cells cultured in the lower chamber were cultured with 1.5 X 107 DNP Fla primed spleen cells for 4 days. Values represent arithmetic mean f the standard error of the mean of 4 cultures.
8
FEZDMANN,
CONE,
AND
MARCHALONIS
tion prior to the binding assay little or no radioactivity was bound to the cells. After correction for losses in washing, at least 705% of the radioactivity precipitated as immunoglobulin was specifically absorbed by macrophages. To determine whether T cell antigen receptors, bound to macrophages in the absence of antigen, could subsequently mediate cell cooperation, AT&,= were cultured for 2 days in the upper chamber of double flasks either with DNP KLH or DNP FyG, and macrophages cultured in the lower chamber. These macrophages were harvested, washed and cultured with DNP Fla primed spleen cells in the presence of antigen, DNP KLH (Table 4). An anti-DNP response occurred with the addition of DNP KLH if ATCRLH were used in vitro, hut not if ATCFVc were cultured. These results indicated that the macrophagcs did bind a T cell product, which enabled them to subsequently bind specific antigen and immunize B cells. However, these macrophages did not induce as high antibody responses as those which bound receptor-antigen complexes. Cooperation
After
Initial
Binding
of Antigen
to T Cells
Cell interaction of T and B lymphocytes across the nucleopore membrane is specific for antigenic determinants which are linked to those recognised by T cells (7, 10). Since the T cell specificity of cooperation was the same as that of T cell activation, the T cell component should have the same specificity as the T cell receptors. To determine whether T cell receptors for antigen could participate in the process of cell cooperation, ATC RLH were first incubated for 1 hr at 4°C with 1 pg/ml DNP KLH, washed thoroughly and then cultured in double chambers with peritoneal exudate cells in the lower compartment, in the absence of further antigen (Table 5). The cooperative capacity of the prcincubated cells was the same as that when antigen was prcscnt for the entire period of initial culture. These results suggested that at least part of the mediator of cell cooperation was by T
TABLE
3
BINDING OF lzSI (iodide) KLH ACTIVATED T CELL PROTEINS TO VARIOUS CELL TYPEF Cell surface protein treatment
lzsI (iodide) Macrophages
Thymus
SURFACE
counts bound to: B cells~
cells
None
1. 1445 f
263
2.
55 f
26
3.
Coprecipitated with antiimmunoglobuliu
4. 33.8 f
33.3
5. 81.6 f
5.1
6.
Coprecipitated NRS
7. 1448 f
307
8.
123 i
53
9. 69.3 f
with
Significance
94 + 15 0
23
1 P 4 I’ 4 7
and 2, 3 or 4 < 0.001 and 7 < 0.001 and 5 P > 0.05 and 8 or 9 P < 0.001
a 100 ~1 of lzsI (iodide)-labeled cell surface proteins were incubated with 2 X lo6 macrophages, thymus cells or B cells in 100 ~1 PBS for 2 hours at 4OC. The cells were washed 3-6 times and radioactivity in the pellet determined. Data are represented as counts per minute. Results represent the arithmetic mean ZIZSE of values obtained in 3 experiments with 3-4 replicates per group in each experiment. Background radioactivity has been subtracted from all values. b B cells = 2 X 106 nu/nu spleen cells.
cm.L
INTERACTIONS
In Vitro TABLE
BY
T
CELL
9
IgM
4
CELL COOPERATION WITH SEQUENTIAL BINDING OF THE T CELL COMPONENT, THEN ANTIGEN TO MACROPHAGES Anti-DNP response AFC/culture
Cells cultured Lower
Upper DNP DNP DNP
ATCKLH ATCKLH AT&G
KLH F,G KI,H
Macrophages Macrophages Macrophages
780 f 105 340 f 956 0
a 2 X 106ATCmn or ATCs-x: were cultured in the upper chamber with either 1 rg/ml or DNP F,G for 2 days with peritoneal exudate cells as a source of macrophages chamber. IO5 peritoneal exudate cells were cultured with 1.5 X 10’ DNP Fla primed for 3 days in the presence of 1 pg/ml DNP KLH. Values represent the arithmetic mean error of 4 cultures. * P < 0.05 compared with group initially cultured with DNP KLH.
cell receptor-antigen, already complexed, absorbed by the peritoneal exudate cells.
DNP KLH in the lower spleen cells f standard
released by the T cells and subsequently
DISCUSSION The experiments presented here were designed to ascertain the chemical nature of the factor released by T cells which participates in specific cooperation with B cells. The cell surfaces of activated T cells used in in vitro cooperative systems (6, 7) were labeled with 1Z51-iodide (12). These were shown to possess surface immunoglobulin with light chains, and heavy chains which resembled p chains when analyzed by polyacrylamide gel electrophoresis in acid urea. The gel electrophoretic mobility of the intact immunoglobulin was consistent with a molecular weight of approximately 180,000 for the protein. These results corresponded well to previous results obtained for surface immunoglobulin of T cells activated to histocompatibility antigens (18). Furthermore, the presence of light chains and TABLE
5
COOPERATION ACROSS A MEMBRANE AFTER INITIAL ANTIGEN TO ATCa Antigen
DNP
KLH
DNP F,G
BINDING UF
Duration
Anti-DNP Response (AFC/culture ZJZSE)
2 days 1 hr 2 days 1 hr
580 f 140 490 f 100 0 0
(i 2 X lo6 ATCKL~ were incubated with 1 rig/ml DNP KLH or DNP,FG for 1 hour at 4”C, washed. To half of each group of cells antigen was readded. The ATCmn were then cultured in double chamber flasks with peritoneal exudate cells in the lower compartment. IO5 macrophages from each group were then cultured for 3 days with DNP Fla primed spleen cells. Values are the arithmetic mean of 4 cultures & the standard error of the mean of 4 cultures, Similar results were obtained in 2 other experiments.
10
FELDMANN,
CONE,
AND
MARCHALONIS
p chains confirmed prior results demonstrating that the factor mediating T cell and B cell interaction in the in vitro systems was specifically inhibited by treatment with rabbit antisers to murine K-chains and p-chains (10). The binding specificity of the isolated surface immunoglobulin for the activating antigen was established by coprecipitation assay (Table 1) . The capacity of the surface IgM of ATC to bind various cell classes was investigated (Table 3) and found to correlate precisely with the binding of the collaborative factor (Table 2). These observations suggested that the T cell component of the collaborative factor resembled the specific receptor of ATC. Evidence to support this concept can be marshalled as follows: (a) AT&H binds sufficient DNP-KLH in 60 minutes at 4°C to collaborate efficiently with B cells, with adherent cells as an intermediate (Table 5) ; (b) metabolic inhibitors abrogate both collaboration by ATC (8) , and the release of surface immunoglobulin (25) ; (c) macrophages bind both T cell surface IgM (Table 3), and the T cell component which, in the presence of antigen, induces antibody responses (Table 2, Ref. 10). The combination of biochemical and tissue culture data presented above is consistent with the hypothesis of Bretscher and Cohn (30) that specific T/B lymphocyte interaction involves “carrier or associative antibody.” We provide evidence that this carrier antibody has a MW of approximately 180,000 and contains p and light chains. The molecule can be described as a monomeric IgM immunoglobulin. However, there is as yet no data on the cell synthesizing the collaborative factor (or T cell monomeric IgM) . The discovery of carrier antibody as one of the T cell components of cell interaction has posed some intriguing questions. Firstly, it is known that B cells possess on their surfaces monomeric IgM immunoglobulin which is probably receptor for antigen (14, 16, 17). Moreover, these molecules turn over in a relatively rapid fashion, compared to T cell IgM immunoglobulin (18, 31, 32). However because B cells have not yet been shown to act as helper cells (33, 34) it is likely that B cell monomeric IgM does not function in cell cooperation. Since monomeric IgM must bind to adherent cells (lo), perhaps macrophages or dendritic cells (35) in order to mediate cell interaction, the inability of B cells to collaborate may be explained by the different binding characteristics of the B cell receptor. Recent experiments indicate that B cell monomeric IgM does not bind to macrophages (38). Secondly, the dual function of the T cell receptor may prove to be of key biological interest. This monomeric IgM not only acts as a receptor for antigen, but also acts as a transmitter of immunological information, extending the range of action of T cells in cooperation. This amplification property of released T cell receptor obviates a major weakness of models of interaction between T cells and B cells which require contact between two rare cell types (36). These two distinct functions of a single receptor molecule are thus far unique in biological systems. ACKNOWLEDGMENTS The technical assistance of Mrs. J. Thompson, Misses B. Pike, J. Jackson, P. Smith J. Gamble is gratefully acknowledged.
and
CELL
INTERACTIONS
I?2
YitfV
BY
T CELL
IgM
11
REFERENCES 1. 2. 3. 4. 5.
Miller, J. F. A. P., and Mitchell, G. F., Trunsplallt. Ziev. 1, 3, 1969. Davies, A. J. S., Transplant. Rev. 1, 43, 1969. Claman, H. N., and Chaperon, E. A., Transplant. Rev. 1, 92, 1969. Mitchison, N. A., Eur. J. Zmmunol. 1, 10, 1971. Cheers, C., Breitner, J. C. S., Little, M., and Miller, J. F. A. P., Nature New Biology 232, 248, 1971. 6. Feldmann, M., and Basten, A., Nature New Biology 237, 13, 1972. 7. Feldmann, M., and Basten, A., J. Exp. Med. 156, 49, 1972. 8. Feldmann, M., and Basten, A., Eur. J. Imrnwzol. 2, 213, 1972. 9. Feldmann, M., J. Exp. Med. 135, 1049, 1972, 10. Feldmann, M., /. Erp. Med. 136, 737, 1972. 11. Marchalonis, J. J,, Biochem. J. 113, 299, 1969. 12. Marchalonis, J. J., Cone, R. E., and Sante, V., Biochem. J. 124, 921, 1971. 13. Phillips, D. R., and Morrison, M., Biochemistry 10, 1766, 1971. 14. Baur, S., Vitetta, E. S., Sher, C. J., Schenkein, I., and Uhr, J. W., J. Zmmunol. 106, 1133, 1971. 15. Marchalonis, J. J., Atwell, J. L., and Cone, R. E., Nature (Londor~) 235, 240, 1972. 16. Marchalonis, J. J., Cone, R. E., and Atwell, J. L., Proc. Nat. Acnd. Sci. U.S.A. (in press), 1972. 17. Vitetta, E. S., Baur, S., and Uhr, J. W., J. Exp. Med. 134, 242, 1971. 18. Marchalonis, J. J., Cone, R. E., Atwell, J. L., and Rolley, R. T., Ztz “The Biochemistry of Gone Expression in Higher Organisms” (in press), 1972. 19. Feldmann, M., Wagner, H., Basten, A., and Holmes, M., Aust. J. Exp. Biol. Med. 56, 651, 1972. 20. Miller, J. F. A. P., and Warner, H. L., Znt. Arch. All. 40, 59, 1971. 21. Feldmann, M., J. Exp. Med. 135, 735, 1972. 22. Cunningham, A. J., and Szenberg, A., Immunology 14, 599, 1968. 23. Shortman, K., Williams, R., Jackson, R., Russell, P., Byrt, P., and Diener, R., /. Cell. Biology
48, 566, 1971.
24. Shortman, K., Williams, N., and Adams, P., J. Zmmunol. Methods (in press), 1972. 25. Cone, R. E., Marchalonis, J. J., and Rolley, R. T., J. Exp. Med. 134, 1373, 1971. 26. Edelman, G. M., and Marchalonis, J. J., In “Methods in Immunology and Immunochemistry (C. A. Williams and H. W. Chase, Eds.), Vol. 1, p. 405. Academic Press, New York, 1967. 27. Parish, C. R., and Marchalonis, J. J., Asal. Biochem. 34, 436, 1970. 28. Philips-Quagliata, Levine, B. B., Quagliata, F., and Uhr, J. W., J. Exp. Med. 133, 589, 1971. 29. Baston, A., Miller, J. ‘F. A. P., Sprent, J., and Pye, J., J. Exp. Med. 135, 610, 1972. 30. Brestcher, P. A., and Cohn, M., Nature (London) 220, 444, 1968. 31. Wilson, J. D., Nossal, G. J. V., and Lewis, H., Eur. 1. Zmnzunol. 2, 225, 1972. 32. Lerner, R. A., McConahey, P. J., Jansen, I., and Dixon, F. J., 1. Exp. Med. 135, 136, 1972. 33. Feldmann, M., Eur. J. Zntmm~tol. 2, 130, 1972. 34. Miller, J. F. A. P., Sprent, J., Basten, A., Warner, N. L., Sreitzer, J. C. S., Rowland, G., Hamilton, J., Silver, H., and Martin, W. J., J. Exp. Med. 134, 1266, 1971. 3.5. Guttman, G., and Weissman, I. L., Z~nmunologg 23, 465, 1972. 36. Mitchison, N. A., Taylor, R., and Rajewsky, K., I?$ “Developmental Aspects of Antibody Formation and Structure” (J. Sterzl, Ed.), p. 517. Publ. House. Czech. Acad. Sci., Prague, 1970. 37. Bailey, N. T. J., “Statistical Methods in Biology” p. 43. English Univ. Press Ltd. London, 1959. 38. Cone, R. E., ‘Feldmann, M., Marchalonis, J. J., and Nossal, G. J. V. Zmmunology (in press), 1973.