Mitogens and T-independent antigens stimulate T lymphocytes to secrete Ia antigens

CELLULAR

IMMUNOLOGY

Mitogens

and

33, 134-144 (1977)

T-Independent Antigens Stimulate to Secrete la Antigens

CHRISTOPHER

R. PARISH

Department of Microbiology, John and Department of Medicine, Received

AND IAN

Curtilz Austin August

T Lymphocytes

F. C. MCKENZIE

School of Medical Research, Canberra, Hospital, Heidelberg, Australia. 30,1976

Previous reports from this laboratory suggest that certain Z region-associated (la) antigens can be detected in normal mouse serum. It was found that, when mitogens are injected into mice, they produce substantial increases (up to 125fold) in the levels of these Ia antigens in mouse serum. Similar increases were obtained when either T- or B-cell mitogens were injected. Furthermore, in vitro and in vivo studies demonstrated that the mitogens stimulated T cells to secrete Ia antigens. It appears likely, however, that the Ia antigens detected in these studies may differ from the conventional Ia glycoproteins found on the surface of B lymphocytes. All T-independent antigens tested also augmented the concentrations of Ia antigen in serum, the increases depending on the T-independent antigen injected and ranging from 3- to 125fold. In contrast, T-dependent antigens, unless injected in large amounts, were unable to produce detectable changes in the serum levels of Ia antigen. These data indicate that an inverse relationship exists between the T dependence of an antigen and its ability to stimulate T cells to secrete Ia antigens. On the basis of this conclusion it is proposed that all antigens are T dependent and merely vary in the efficiency with which they activate T cells to release helper factors.

INTRODUCTION The I region-associated (la)’ antigens of mice are coded for by a group of genes located within the H-2 complex and exhibit a remarkable polymorphism ( 1, 2). The biological function of the Ia antigens is uncertain at this time although they may represent products of the immune-response (Ir) genes. Recently it was reported that substantial amounts of Ia antigen can be detected in normal mouse serum (3, 4). Although this serum-borne Ia antigen is associated with high-density lipoproteins (4, S), the antigenic material is of low molecular weight and can be dissociated from the lipoproteins by organic solvents or by very high or very low salt concentrations (5, 6). In fact, a range of chemical analyses has suggested that the low molecular weight Ia antigen contains an oligosaccharide moiety (6) and may constitute a ganglioside (5). 1 Abbreviations used : Ia, Z region-associated ; PHA, phytohemagglutinin ; Con A, concanavalin A ; LPS, lipopolysaccharide; PPD, purified protein derivative; DXS, dextran sulphate; PWM, pokeweed mitogen; POL, polymerized flagellin; MON, monomeric flagellin; PB, polymyxin B; S3, pneumococcal polysaccharide type S3; PVP, polyvinyl pyrrolidone; BSA, bovine serum albumin; FGG, fowl +y-globulin; HGG, human y-globulin; SRBC; sheep red poly-L- (Tyr,Glu)-poly-nr.-Alablood cells ; HRBC, horse red blood cells ; ( (T,G)-A-L), poly-L-Lys ; PBS, phosphate-buffered saline ; FCS, foetal calf serum. 134 Copyright 1wl rights

Q 1977 by Academic Press, of reproduction in any form

Inc. reserved.

ISSN 0008-8749

SECRETION

OF

Ia

ANTIGENS

BY

T

CELLS

135

Additional studies have demonstrated that the serum la antigen is secreted bq long-lived recirculating T cells which have been recently activated by antigen (7). During the course of these studies it became apparent that certain substances, when injected into mice, produced large increases in the levels of Ia antigen in serum (7). This phenomenon is examined in more detail in this paper, and it is demonstrated that mitogens and T-independent antigens very effectively stimulate T ~~11s to secrete Ia antigens, whereas T-dependent antigens are mucll weaker stimhtors of Ia secretion. MATERIALS

AND

METHODS

An&&. Inbred CBA/H, A.TL, and A.TH mice of either sex and from 6 to 12 weeks of age were used as cell and serum donors. Athymic nude nw/nlf mice were bred with a CBA/H background. Antisera. A xenogeneic anti-Ia serum was raised in rabbits against the la antigens expressed by cells from CBA/H mice (i.e., potentially Ia specificities : 1, 2, 3, 7, 15, 17, 18, 19, and 22). The preparation of this antiserum has been described in detail previously (3) and involved immunizing rabbits with CBA/H serum ant1 then absorbing the resultant antiserum with exhaustively dialysed CBA/I-I seruln. An allogeneic anti-Ia serum was raised in A.TH mice against A.TI, spleen, thymus, and lymph node cells as reported previously (7) and should detect la specificities 1, 2, 3, 7, 15, 19, and 22. Similarly, an A.TL anti-A.TH serum was raised which should detect Ia specificities 4, 5, 9, and 12. The binding of the xenogenic anti-Ia antibodies to mouse leukocytes was detected by a two-stage rosetting procedure (3), whereas the reaction of mouse antibodies \vith leukocytes was assessedby cytotoxicity (3). Mitogens and [email protected]. Phytohemagglutinin (I’HA) was purchased from Wellcome Laboratories, Beckenham, England, and pokeweed mitogen ( PM’M ) was ol)tained from Grand Island Biological Co., Grand Island, N.Y. Jack bean coilcanavalin A (Con A), crystalline egg white lysozyme (Grade VI). and crystalline ovalbumin (grade V) were supplied by Sigma Chemical Co., St. Louis, MO. Dextran sulphate (DXS) (MW 500,000) and Ficoll 400 were obtained from Pharmacia Fine Chemicals, Uppsala, Sweden. Lipopolysaccharide B (LPS) isolated front Esclzcviclzia coli 0 128: Bl2 was purchased from Difco, Detroit, Mich., and polyvinylpyrrolidone K90 (PVP) was provided by Fluka, Switzerland. Hunlan y-globulin (HGG) fraction II, tuberculin purified protein derivative (PPD), horse red blood cells (HRBC), and sheepred blood cells (SRBC) were purchased from Conlmonwealth Serum Laboratories, Melbourne, Australia. Fobvl y-globulin ( F( ;G ) was kindly provided by Dr. P. Bretscher of this department and was purified 1)~ ~IIImonium sulphate precipitation and ion-exchange chromatography. Crystalline l)ovitle serum albumin (BSA ; A grade) was obtained from Calbiochem, San Diego. Calif.. and polymyxin B sulphate (PB) was supplied by Pfizer Chemicals, I’arr;lmatta, New South IVales, Australia. Pneuniococcal polysaccharide type S3 (S3) was kindly supplied by Dr. J. Humphrey, National Institute for 11IedicalKcsearch, Mill Hill, London, and the synthetic branched polypeptitlc antigen poly-1*(Tyr, Glu)-poly DL-Ala-poly-L-Lys [ (T,G) -A-L j 1 was kindly donated by Dr. G. Roelants, Base1Institute of Immunology, Switzerland. Hemocyanin, a pentamer of molecular weight 150,000, was crystallized front tile haemolymph of the South Australian crayfish, Juslls lalandii, as previously tle-

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scribed (8). Monomeric flagellin (MON) and polymerized flagellin (POL) were obtained from S. typhi+nurium (strain SL870 ; H antigen 1,2) and were prepared as reported earlier (9). A stable complex between LPS and PB was produced by carrying out the incubation procedure described by Jacobs and Morrison (10). The various mitogens and antigens were suspended in phosphate-buffered saline (PBS), and either 0.2 ml was injected intraperitoneally or 0.5 ml was injected intravenously into mice. Assays for la antigens. The relative amounts of Ia antigen in sera and culture supernatants were estimated by two different antigen-antibody inhibition assays which have been reported earlier (3). The first assay measured the ability of serial dilutions of the sample to neutralise the cytotoxicity of the allogeneic anti-Ia serum. An antibody dilution was chosen for the inhibition assays which was one tube less than the highest dilution causing maximum cell lysis. Fifty-microliter doubling dilutions of each sample were prepared in L15 tissue culture medium (Microbiological Associates) containing 5% BSA, and then 50 ~1 of the appropriate dilution of anti-Ia serum was added to each well. After incubation at 4°C for 18-24 hr spleen cells of the appropriate strain were added to each tube, and the cytotoxicity test was performed as previously described (3). With the A.TH anti-A.TL serum, A.TL or CBA/H target cells were used, whereas, with the A.TL anti-A.TH serum, A.TH target splenocytes were employed. The second inhibition assay assessed the capacity of samples to inhibit the binding of xenogeneic anti-Ia antibodies to lymphocytes, the xenogeneic antibodies bound being detected by a rosetting procedure (3). Initially, a dilution of the xenogeneic antiserum was chosen which was one tube less than the highest dilution which produced maximum rosetting. Fifty microliters of this antibody dilution was added to serial dilutions (50 pl) of the various samples, and the mixture was incubated at 4°C for 18-24 hr. One hundred microliters of CBA/H spleen cells [2 X 10T/ml in PBS-lo% foetal calf serum (FCS)] was then added to each tube, and the rosetting test was performed as previously described (3). In both inhibition assays, inhibitory activity was expressed as the inhibitory activity per milliliter of sample and represented the reciprocal titre required for 50% inhibition of cytotoxicity or rosetting. Preparation of cell suspensions. Spleen cell suspensions were prepared as described in an earlier paper (3). In some cases the spleen cell preparations were treated with anti-Thy-l antiserum and complement (11). The spleen cells were separated into Ig+ and Ig- subpopulations by a previously reported Isopaque/ Ficoll fractionation technique (12). In vitro culture conditions. Spleen cell populations were cultured in vitro as published previously (7). Briefly, spleen cells were cultured at 37°C for 5 hr in tissue culture medium lacking FCS. The culture supernatants were then harvested, dialysed in acetylated dialysis tubing to remove salt, and concentrated by lyophilisation. The resulting concentrate was then assayed for its Ia content by the two inhibition tests. RESULTS Effect of Diflerent

Antigens

and Mitpgens

on the Levels of Ia Antigen

in Serum

A series of earlier reports (3-7) established that substantial levels of Ia antigen are present in normal mouse serum. This serum-borne Ia antigen was readily de-

SECRETION

OF

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ANTIGENS

137

HY T CELLS

I

0

i,

4

2 TIME

[DAYS]

40960

20480

10240

640

320

0

2

4

TIME FIG. 1. Content of Ia antigen in the sera of CBA/H with saline (A), PHA (A), LPS (o), or Con A injected. (a) Ia antigens assayed by allogeneic anti-la geneic anti-Ia serum.

b

[DAYS J

mice at various times after ip injection (a). A SO-fig dose of each mitogcn was serum (1)) Ia antigens assayed by xeno-

tected by both xenogeneic and allogeneic anti-Ia sera (3, 5, 7). On the basis of these findings, experiments were carried out to determine whether injections of different antigens and mitogens influenced the levels of Ia antigen detected ill mouse serum. Initially, groups of CBA/H mice (three to five per group) were injected ip with either saline, Con A (50 pg), PHA (SO pg), or LPS (50 ,ug), and, 2, 4, ant1 6 days after injection, serum samples were collected from the members of each group. pooled, and tested for Ia content by the two inhibition assays. Figure 1, a and b,

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depict the relative levels of Ia antigen detected in the serum of these mice by the allogeneic and xenogeneic anti-Ia sera, respectively. It can be seen that all three mitogens produced a substantial increase in the levels af Ia antigen detected in mouse serum. In the case of PHA and LPS the increase was as high as 50- to lOOfold, whereas Con A produced a more moderate S fold increase. Peak Ia levels were reached 4 days after the injection of each mitogen and tended to decline by Day 6. Both Ia assays gave comparable results. A striking feature of this experiment is that, although the three mitogens tested are mitogenic for different subpopulations of lymphocytes, they all produced increased levels of serum Ia. Several other established mitogens were, therefore, tested to determine whether they had similar effects on the Ia levels in serum. Table 1 presents the relative Ia levels in the serum of mice 4 days after injection of each mitogen, i.e., the day when peak Ia levels were reached. All six mitogens tested augmented the Ia concentrations in serum, the increases depending on the mitogen injected and ranging from 8- to 125-fold. Both T- and B-cell mitogens produced similar effects, and both Ia assays gave similar results. A range of conventional antigens was then tested for ability to elevate the levels of Ia antigen in mouse serum (Table 2). Doses of the antigens which are known to be immunogenic in mice were injected. At this stage it should be emphasised that the division between mitogens and T-independent antigens in this paper is somewhat arbitrary (27). It was found that all five T-independent antigens examined produced a detectable increase in serum Ia levels. The increases, however, varied considerably from antigen to antigen. Thus, POL produced a lOO- to 125fold increase, whereas S3 and PVP stimulated only a 2- to 4-fold rise. The smaller increases are genuine as they were reproducibly detected by both Ia assays. It is noteworthy that LPS, following conjugation with polymyxin sulphate (PB), still elevated very effectively the Ia levels in serum, even though PB is believed to convert LPS to a T-independent antigen which lacks mitogenicity (10). In contrast TABLE Ability

Mitogena

Nil PHA Con A LPS PPD DXS PWM

Dose

.50 50 50 50 1 0.5

a rg rg erg w ml

of Different la Antigens

1

Mitogens to Increase the Levels in CBA/H Mouse Serum

Inhibition

of anti-Ia

serab

Xenogeneic

Allogeneic

320

320

20,480 2,560 32,000 5,120 40,960 10,240

20,480 2,560 40,960 2,560 10,240 5,120

of

Mitogenic target cell T T B B B T,B

Ref.

(I3J4) (13,14) (13,14) (13) (13) (13,14)

0 Mice were injected ip with mitogens 4 days prior to serum sampling. PHA, Con A, and LPS data from Fig. 1. * Both anti-Ia sera directed against Ik antigens (e.g., la 1, 2,3, 7, and 15). The xenogeneic antiserum was produced by immunizing rabbits with CBA/H mouse serum, and the allogeneic antiserum was raised in A.TH mice against A.TL lymphoid cells. Results expressed as inhibitory activity per milliliter of serum and represent the reciprocal titre for 50% inhibition of binding of the anti-Ja sera.

SECRETION

OF

ra

ANTIGENS

TAI3LE Ability

of a Range

BY

1

2

of T-Dependent and T-Independent the Levels of Ia Antigens in Serum

Antigens

--___ Antigen”

Dose

Inhibition Senogcnric

1.10

(‘ELLS

of anti-In

sernh

to Inrreasc I_--. Antigen T dependent

Ref.

Allogeneic

.__-

I’VP

50 50 1 100 100

SRBC MON Ilemorynnin MA FGG HGG ((T,G)-A-L) 1 .ysozyme Ovnlbumin

.50 P#T 1 mg 1 w 100 fig 1 mg loo& 1 mg 1 w

Nil POL LPS + Firoll s3

PR

320 32,000

Pg P!z

20,480 1,920

w N /JR

960

108

C Slice were injected ip with h Anti-la sera and inhibition

2,560 1,2x0

960

6-w

320

320

320

320

240 240 320

HO 200 4X0

320 160

320 320

160

320 320

240

antigen assays

_

320

40,960 5,120

4 days prior to serum as in Table 1.

(15) 110) (IO) (17) (1x1 (1Y) 115) (20)

121) (2.1)

(22) (21) (25) f2oj sampling.

to the T-independent antigens, all the T-dependent antigens tested producetl no detectable increase in the serum Ia levels. Natwr of Inhibitory Mat&al

in Senm

Although there is ample evidence that normal mouse serum contains la antigen (3-7) it could be argued that mitogens and T-independent antigens stimulate the release into serum of substanceswhich are not Ia antigen but which nonspecifically inhibit the Ia antigen assays. In order to test this possibility two mouse strains, namely, A.TH (I”) and A.TL( I”), which are congenic at the I region, were injected with a series of mitogens and the specificity of the inhibitory material in their sera was determined. Groups of either A.TL or A.TH mice (three per group) were injected ip with either saline, POL, Con A, DXS, or LPS, and. 3 days after injection, serum samples were collected from the members of each group and were pooled. The ability, of these sertmi samplesto inhibit the binding of either xenogeneic or allogeneic antiIa antibodies to lymphocytes was then determined. It was found that, with all serum samples, there was a high degree of haplotype specificity in the inhibition observed (Table 3). Thus, serum from normal and mitogen-injected A.TH( I”) mice strongly inhibited the binding of anti-I”, but not anti-I” antibodies to Iymphocytes, whereas serum from A.TL(I”) mice only inhibited the interaction of antiIii antibodies with lymphocytes. This haplotype specificity of inhibition was observed with both xenogeneic and allogeneic anti-Ia sera, although, with the xenogeneic anti-I” antibodies, seru111from A.TH (I”) mice exhibited weak inhibitory activity, i.e., approximately l-59) of the inhibitory activity of A.T12( I”)

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serum. This weak inhibition may be due to shared Ia antigenic specificities between A.TH and A.TL mice (specificities 17 and 18) which the rabbit anti-Ik serum is known to recognize (unpublished data). These specificities indicate that our inhibition assays are specific and are indeed measuring elevated levels of Ia antigen in the serum of mitogen-injected mice. Origin

of la Antigens

in Serum

In an earlier publication from this laboratory (7), it was reported that the Ia antigen in normal mouse serum was derived from long-lived recirculating T lymphocytes. It was important, therefore, to confirm that the high levels of Ia antigen in the serum of mitogen-stimulated mice was also derived from T lymphocytes. The experimental approach was to inject mice iv with mitogen, harvest the spleen cells 4 days later, and then assess the ability of subpopulations of these splenocytes to secrete Ia antigen in vitro. In the experiments depicted in Table 4 the various subpopulations of spleen cells were cultured for 5 hr at 37°C in tissue culture medium lacking FCS. The culture supernatants were then collected, concentrated, and assayed for their Ia content by either the xenogeneic or allogeneic anti-Ia sera. Initially, it was found that spleen cells from LPS-, DXS-, and PHA-injected mice secreted Ia antigens at a much higher rate than did spleen cells from normal mice (Table 4). These secretion rates closely resemble the serum levels of Ia antigen in these mice (see Table 1). However, it was discovered that, with both normal and mitogen-stimulated spleen, the Ia antigen was produced almost exclusively 3

TABLE I-Region

Mitogen”

Origin

of the Inhibitory Mitogen-Injected

Strain donating serum

Material Mice

in the Serum

Inhibition

of anti-Ia

Allogeneic Anti-Ik

of

serab Xenogeneic Anti-Ik

Anti-IB

Nil POL Con A DXS LPS

A.TH A.TH A.TH A.TH A.TH

0 0 0 0 0

60 320 320 120 120

0 40 40 40 20

Nil POL Con A DXS LPS

A.TL A.TL A.TL A.TL A.TL

160 2.560 1000 1280 N.T.”

0 0 0 0 0

80 1280 2560 2560 1920

a Mice were injected ip with mitogens 4 days prior to serum sampling. Doses of mitogens as in Table 1. b Allogeneic anti-P: A.TH anti-A.TL serum and A.TL target cells. Allogeneic anti-In: A.TL anti-A.TH serum and A.TH target cells. Xenogeneic anti-Ik: rabbit anti-CBA/H mouse serum and CBA/H target cells. Results expressed as inhibitory activity per milliliter of serum and represent the reciprocal titre for 50% inhibition of binding of the anti-Ia sera. c N.T., not tested.

SECRETION

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ANTIGENS

TABLE Ability

Spleenfl cells

of Different

Subpopulations Cells to Secrete

Anti-Ia serum

BY

T

111

c‘l:.Ll.S

4

of Normal or Mitogen-Stimulated Ia Antigens in ‘Vitro ___ Ia inhibition

titrc/ml

Spleen

_

of supernatanth --

Unfractionated spleen

Ig-

Ig”

Anti-thy-l treated

___,Normnl

Xenogeneic Allogeneic

160 120

240 140

10 10

10 0

LPS

Xenogeneic Allogeneic

1,280 640

1,280 640

40 40

20 0

I)SS

Xenogeneic Allogeneic

10,240 5,120

10,240 4,000

80 60

160 00;

PHA

Xenogeneic Allogeneic

2,560 1,280

2,560 1,280

40 0

20 0

(1 Donor * Anti-Ia

mice were injected sera and inhibition

iv with mitogens 4 days prior assays as in Table 1.

to sacrifice.

Mitogrn

doses as in Table

1.

*. ‘.._‘.“; .

./

FK. 2. Effect of different mitogens on the levels of Ia antigen in the serum mice or CBA/H ~zu/nu mice. A .50-pg dose of either PHA ( 0, 0) or LPS jected ip on Day 0 into each mouse. The closed symbols represent normal open symbols, CBA/H nz@u mice. The Ia levels were assessed by the inhibition assay. Comparable results were obtained with the allogeneic anti-Ta

of normal CBA/H (A, n ) was inCBA/H mice, the xenogeneic anti-la inhibition assay.

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of Different

Immunization

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MCKENZIE

TABLE

5

Schedules

Immunization schedule”

of SRBC Days

after

3 Nil 109 lo9 109 109 lo9

SRBC SRBC SRBC, SRBC, SRBC,

then then then

109 SRBC 109 SRBC 109 SRBC

320b 160 1,280 400 800 160

to Stimulate

Ia Secretion

last injection 6 320 240 1,280 320 800 640

10 320 640 640 320 320 320

a Groups of CBA/H mice (three per group) were injected iv with the SRBC and there was a gap of 11 days between the first and second doses of SRBC. b Ia inhibition titre per milliliter of serum as measured by the allogeneic anti-Ia serum.

by an Ig-, Thy 1’ cell, i.e., a T cell. Almost identical results were obtained with the two assays for Ia antigen. An additional method of demonstrating that mitogens stimulate T cells to secrete Ia was to assess the levels of Ia antigen in the serum of congenitally athymic nu/nu mice following injection of mitogens (Fig. 2). It was initially confirmed that nu/nu mice have very low levels of Ia antigen in their serum (< 5% that of normal mice) (7). Following injection of either PHA or LPS there was a substantial increase (up to 20-fold) in the levels of Ia antigen in the serum of nu/nu mice (Fig. 2). However, even after such increases, the concentration of Ia in the nu/nu serum was < 2% of that detected in the serum of mitogen-stimulated normal mice. This result is consistent with the T-cell origin of serum Ia antigens. It has already been reported (11) that nu/nu mice, similar to the ones used in our experiments, contain a small pool of functional T cells. Presumably, these T cells produce the low levels of Ia antigen detected in nu/nu mouse serum. Can T-Dependent Antigens Stimulate la Secretion?

In Table 2 it was demonstrated that a single immunogenic dose of a T-independent antigen substantially elevated the serum levels o’f Ia antigen, whereas a similar injection of T-dependent antigen had no detectable effect on the Ia levels in serum. Thus, experiments were carried out to determine whether a T-dependent antigen, when injected in high and/or multiple doses, could augment the serum Ia levels. It was found that a low dose (i.e., lo6 cells) of a T-dependent antigen, SRBC, produced no detectable change in serum Ia, whereas a high dose (i.e., lo9 cells) resulted in a threefold increase in Ia levels 3 and 6 days after injection (Table 5). This increase, although small, was genuine, as the sameresult was obtained in three consecutive assays. Similarly, repeated low dosesof SRBC had no effect on serum Ia levels, whereas repeated high doses maintained slightly augmented levels of Ia. Similar results were obtained when HRBC were the antigen. Thus, it appears that under certain immunization conditions T-dependent antigens can elevate the concentrations of Ia antigen in mouse serum.

SECRETION

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RY

T CELLS

14.1

DISCUSSION We have demonstrated in this paper that mitogens and T-independent antigens efficiently stimulate T lymphocytes to secrete Ia antigen. In contrast, even when injected in high doses, T-dependent antigens are much weaker stimulators of Ia secretion. Thus, there seems to be an inverse relationship between the T tlepentlenq of an antigen and its ability to stimulate Ia secretion by T cells. At this point it should be emphasised that the putative “Ia antigen” we detect in mouse serum may be controlled by genes in the I region which are distinct from the In genes which control the Ia glycoproteins predominantly expressed on 1: lymphocytes. This is a necessary qualification as evidence is mounting which suggests that the antigenicity of the Ia glycoproteins isolated from the surface of 1: cells is protein, rather than carbohydrate, in nature (2s j. On the other hand, it appears likely that the Ia antigens we detect in nlouse serum are related to the 1:~ antigens carried by T cell-derived helper (29) and suppressor (30) factors. It has been proposed for several years that “two signals” are required for tile induction of an antibody response (3 1, 32). The first signal is mediated by antigen binding to Ig receptors on antigen-specific 13 cells, and the second signal is delivered by antigen-activated helper T cells. Consistent with this theory is the ()I)servation that antigen-activated T cells release both nonspecific (33, 34) and antigen-specific (29, 35) factors which appear to be able to deliver “signal 2” to I; cells. In most cases these factors are ~~o~~in~tnunoglol,ulin in nature ant1 carry la antigens (29, 36). It seems likely, therefore, that the low nlolecular weight Ia antigen which we detect in mouse serum is in some way related to these T cell-rq)lacing factors. Such a proposal is supported by our recent observation that the T cells which secrete Ia antigen bear the antigenic markers of helper T cells, i.e., I,yl+:! la (37). \lVhen these data are coupled with our observation tllat there seems to I)e an inverse relationship between the T dependency of an antigen and its ability to stimulate T cells to secrete Ia antigens, a reasonable conclusion is that T-intlcpentlent antigens do not exist as such. Antigens merely vary in their capacity to recruit T help, apparent T-independent antigens being the most efficient activators of ‘I‘ help. Of course, why antigens differ in their ability to recruit help remains to be established. Perhaps the most intriguing finding in this paper is the observation that mitogrns, irrespective of their mitogenic target, very efficiently stimulate T cells to secrete Ia antigens. This finding suggests that T cells (perhaps memory 7‘ cells) require little or no cell division after mitogen stimulation before they start to secrete Ia antigens. Also, these results again raise the question of whether some form of T help is involved in mitogenic responses (13, 14). Presumably, the target cell of mitogcnesis is dependent upon the surface binding properties of the mitogen. ACKNOWLEDGMENTS We are grateful to Ms. Aira Chilcott for expert technical ported in part by a contract obtained from the National Cancer Health (No. l-CB-32925) to I. McKenzie and by grants from tion and the National Health and Medical Research Council.

assistance. The work was supInstitute, Naional Institutes of the Tobacco Research t’ounda-

REFERENCES 1. Shrcffler, D. C., and David, C. S., Adzmt. I~~z~rzwzol. 2. David, C. S., Transfilant. Rev. 30, 299, 1976.

20, 125, 1975.

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