Immunology 1977 33 321

Non-specific factor replaces T cells in an IgG response to soluble antigens J. R. NORTH, J. T. KEMSHEAD & B. A. ASKONAS National Institute for Medical Research, The Ridgeway, Mill Hill, London

Received 18 November 1976; acceptedfor publication 13 January 1977

(KLH) primed cells leads to B-cell division and maturation resulting in the appearance of IgM and IgG anti-DNP antibody secreting cells. Considerable attention has been focussed on the nature of trigger(s) or signal(s) which must be delivered to a B cell in order to initiate this sequence (reviewed by Bretscher 1975; Moller, 1975). However, less consideration has been given to the possible need for continuation of such stimuli in order that activated B-memory cells might complete their development into IgG secreting cells. Since we showed previously that the B cells giving rise to an IgG response in 5-day MishellDutton cultures of DNP-KLH primed and boosted (p/b) mouse spleen cells are already intermediates on the pathway to IgG secretion (North & Askonas, 1976), while still requiring extensive proliferation (Kemshead, North & Askonas, 1977) such cultures offer the opportunity to investigate antigen and T-cell requirements for IgG response at late points in this sequence of

Summary. The antigen and T-cell requirements for the final stages of proliferation and maturation of DNP-KLH primed and boosted mouse spleen cells into IgG antibody secreting cells have been studied in vitro. The requirement for free antigen ceases after 24-48 h in vitro. The carrier-specific T-cell requirement for triggering of activated B cells by a soluble antigen (DNPKLH) can be replaced in T cell-depleted cultures by non-antigen specific T cell-replacing factors (TRF). However, if the carrier protein is changed, TRF restores the IgG response of T cell-depleted cultures only if antigen is presented to B cells in particulate form, e.g. on the surface of macrophages, or in the presence of small amounts of antibody against the carrier protein. Thus, direct interaction between soluble protein and B cells is not sufficient to allow TRF to effectively replace specific T cells. Since TRF must be added at the start of culture, the initiation of B-cell maturation into IgG secretion by TRF occurs during B-cell proliferation, and is followed by further proliferation before IgG antibody can be detected.

events.

Although the replacement of specific T cells by a non-antigen specific T cell-derived factor has been readily achieved in vitro for IgM responses (Schimpl & Wecker, 1972 a, b; Dutton, 1975; Waldmann, 1975) restoration of IgG responses has been less reproducible and less effective (Schimpl & Wecker, 1973; Henry, 1975). We show here that such a factor can effectively replace specific T cells in the late stages of B-memory cell activation into IgG secretion only when the antigen possesses certain physical characteristics.

INTRODUCTION Interaction of an antigen, DNP-KLH, with DNP primed B memory cells in the presence of carrier Correspondence: Dr B.A. Askonas, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 IAA.

321

322

J. R. North, J. T. Kemshead & B. A. Askonas MATERIALS AND METHODS

Mice, tissue culture, antibody-forming cell assays CBA/Ca mice were primed using 100 jpg dinitrophenylated keyhole limpet haemocyanin (DNPKLH) or dinitrophenylated chicken globulin (DNPCG) with alum and Pertussis 2-4 months before a boost with 10 pig DNP-KLH or DNP-CG (North and Askonas, 1976). Seven days later, spleen cell suspensions were prepared and treated with phosphate-buffered saline (PBS*) and 5% FCS to promote dead cell agglutination (Parish, Kirov, Bowern & Blanden, 1974). Agglutinates were gently centrifuged out before culture by the method of Mishell & Dutton (1967) for 5 days with 001 pg/ml DNP9,KLH or 0 01 jg/ml DNPI7-CG. Indirect anti-TNP plaque-forming cells (PFC) were enumerated by a plaque assay on microscope slides (Dresser & Greaves, 1973) using TNP1oHRBC (Rittenberg & Pratt, 1969; North & Askonas, 1976) and a polyvalent rabbit anti-serum to mouse immunoglobulins. Indirect PFC in this system have been shown to be largely yG1, although minor proportions of yG2a, yG2b and yA antibody secreting cells are also detected (North and Dresser unpublished observation). T cell depletion of spleen cell suspensions A goat antiserum to mouse brain (Golub, 1971) was absorbed three times with CBA mouse liver cell membranes and twice with either 2 5 x 107 CBA bone marrow cells or 2 5 x 106 nu nu (Laboratory Animals Centre, Carshalton) spleen cells per ml of serum at a 1/3 dilution. The resulting antiserum killed 95-99% of CBA thymocytes, 0-3% bone marrow cells and 35-40% of CBA spleen cells, assessed by Eosin exclusion. Agarose and mouse red cell-absorbed rabbit serum was used as a source of complement. Bulk treatment of spleen cells before culture was in RPMI 1640 medium with 5Y% foetal calf serum (FCS), at a cell concentration of 3 3 x 107 cells/ml for 45 min at 370. After incubation, cells were washed three times with PBS + 5% FCS and agglutinated dead cells removed (Parish et al., 1974) so that 8595% of the remainder were still viable.

Thymus replacing factor (TRF) This was prepared following the method of Schimpl & Wecker (1972a). Mixtures of CBA/Ca spleen cells * The PBS, pH 7-4 for this purpose was made to the following recipe: KH2PO4-2 g/l; Na2HPO4- 856 g/l; NaCl-4-5 g/l

and either DBA/2 or C57 BI/6 spleen cells at a total of 10' cells/ml were incubated in 60 mm petri dishes (Falcon Plastics) on a rocking platform at 370 for 24-26 h in RPMI 1640 and 1 % FCS. The medium was centrifuged at 350 g and then 3200 g in the cold to remove cells and cell debris. After millipore filtration, aliquots were stored at - 700. The concentration of FCS was increased to 5% before use. Washing of Mishell-Dutton cultures Where the culture medium was changed during the experiment, medium and non-adherent cells were gently removed from each dish and centrifuged at room temperature at 320 g for 5 min. Cells were resuspended in fresh tissue culture medium and returned to the same culture dishes.

Antibody to KLH CBA mice were primed with 100,ug KLH with alum and Pertussis, and 2-4 months later boosted with 10 pg KLH. Ig in a serum pool (taken 10-18 days after boosting) was precipitated with 50% saturated ammonium sulphate, and dialysed against saline and finally Hanks's solution before millipore filtration and storage at -20°. The stock solution contained about 250 pg anti-KLH antibody per ml measured by precipitation assay.

DNP-KLH-Sepharose 0 4 g dry weight of activated Sepharose 4B (Pharmacia) was incubated at room temperature in 0-1 M borate buffer for 2-4 h, with 200 ug DNP9 I-KLH (Feldmann, Greaves, Parker & Rittenberg, 1974). Unreacted groups were blocked with 1 M ethanolamine and the conjugated beads washed with cycles of pH 4 and pH 8 buffers. Beads were stored in borate-buffered saline pH 8 4 and washed extensively in PBS and culture medium before use. An estimate of 3-2 x 106 beads per ml of settled Sepharose was made by counting in a haemocytometer chamber.

Antigen-bearing macrophages Cells were washed from the peritoneum of 6-12 month-old CBA mice with PBS + 0 02 % heparin and centrifuged at 40 in plastic tubes. After washing, the cells were incubated on ice at 2-4 x 106 cells/ml with DNP91-KLH at 3 ug/ml for 30 min. Unbound antigen was removed by multiple centrifugations in the cold, and 3-5 x 105 cells were added to MishellDutton cultures of 107 spleen cells/ml.

T cells in an IgG response to soluble antigens RESULTS Dependence of IgG response on T cells Treatment of DNP-KLH or DNP-CG primed and boosted (p/b) spleen cells with either AKR anti-Thy 1:2 or a goat anti-T-cell serum and complement ablated their ability to mount an IgG anti DNP response in vitro (Table 1). Since viable cell numbers per culture were maintained constant, these cultures contained about twice as many non-T cells as untreated cultures. The response per input B cell was therefore reduced to approximately 5 % of the control value in the experiment shown. Addition of carrierprimed spleen cells restored the response, but not when depleted of T cells. A suboptimal concentration of goat anti-T serum

323

and complement often increased the IgG response more than expected on the basis of the input of B cells per culture. This suggests the presence of suppressive T cells in the spleens of primed and boosted mice which are relatively more sensitive to antiserum treatment than helper cells. Restoration ofIgG response by aliogeneic supernatants Twenty-four-hour supernatants of mixed cultures of DBA/2 and CBA/Ca spleen cells (TRF, Schimpl & Wecker, 1972a) restored the IgG response ofDNPKLH primed and boosted cells after T-cell depletion and in some instances, higher levels of IgG PFC were induced than in cultures of untreated cells (Table 2). Supernatants from cultures of either CBA

Table 1. Dependence of IgG response upon T cells

Treatment of cultured cells*

IgG PFC/culture on day 5

DNP-KLH cellst

KLH cellst

1I0' untreated 107 C' treated 107 G a T treated 3 x 106 untreated 7x 106 G a T treated 3 x 106 G x T treated 7 x 106 G a T treated *

All

30,728 46,124 3150 14,782 2545

(s.e.) (1-04)

(1[13) (1-08) (I 09) (-)

cultures shown contained 0 01 ug/ml DNP-KLH.

t Donor mice primed and then boosted 7 days before killing. Table 2. Restoration of IgG response by TRF in T-depleted cultures

IgG PFC/culture day 5 (s.e.)

Treatment* of DNP-KLH cells

107 Untreated 107 G

a

T and C'

107 G a T and C' 0-5 ml TRF 107 G a T and C' and 0 5 ml DBA/2 supernatant 107 G a T andC' and 0 5 ml CBA/Ca supernatant

001 pg DNP-KLH

Expt 1

+ + + + -

9785 (1[09) 15 (4 70) 26 (3 60) n.d. 15,017 (1-47) n.d. n.d. n.d.

+ -

n.d. n.d.

Expt 2

9300 0 1733 54 35,294 50 251 0

(-)

(1-12) (5 20)

(1[19) (2 96) (2 28)

7 (3 60) 0

n.d. = Not done. * Donor mice primed and then boosted 7 days before killing. D

Expt 3 25,191 (1-08) 20 (10) 385 (1-29) n.d. 10,966 (1-07) n.d. n.d. n.d.

n.d. n.d.

I

.-

6000

I

,

I

to0

-0 cLO

"IA-1

4000 _

0 cr IL

(0

0W a,

200C I/

I// 0

0-2 Volume of TRF (ml)

0-4

Figure 1. Titration of TRF in T cell-depleted cultures. (@) DNP-KLH p/b spleen cells cultured with 0 01 pg/ml DNPKLH; (x) 107 DNP-KLH p/b cells, after treatment with goat anti-T serum and complement, were cultured with 0 01 pg/ml DNP-KLH for 5 days. Different volumes of TRF (24-h supernatant of allogeneic cell cultures) were added at time zero.

goat anti-T serum and complement, cultured with 0 5 ml TRF and 0 01 pug DNP-KLH/ml.

or DBA/2 spleen cells alone did not have this activity. The response obtained was markedly dependent on TRF concentration (Fig. 1) and on TRF addition from time zero (Fig. 2). A delay in addition markedly reduced the IgG response seen at day 5. The appearance of PFC in T cell-depleted cultures in which TRF was present throughout followed kinetics indistinguishable from those in cultures of untreated cells

10,000

to

Days of culture

Figure 3. Kinetics of IgG response in presence of TRF. (0) 107 DNP-KLH p/b cells incubated with 0 01 pg/ml DNPKLH/ml; (A) 107 DNP-KLH p/b cells after treatment with

8000\

(Fig. 3). k

0

Removal of TRF supernatant on day 1 of culture still resulted in full restoration of the IgG response (Table 3). This result may reflect either a transient need for TRF during the maturation of an intermediate B cell or may be due to absorption of the active components to activated B cells (Schimpl & Wecker, 1972b) or accessory cells in the cultures, thus making the factor available for several days.

\

o

IL a-\

\

0

Day of treatment

Figure 2. Timing of TRF addition. (-) DNP-KLH p/b spleen cells cultured with 0 01 ug/ml DNP-KLH; (x) cultures of G- anti T-treated cells given TRF at time zero and washed at times indicated, TRF then replaced; (A) cultures of G- anti T-treated cells without TRF at time zero, TRF added after washing at times indicated; (e) cultures of G- anti T-treated cells in the absence of TRF.

Restoration of IgG response to a heterologous haptencarrier protein We wished to test whether TRF replaces all functions of T cells required during the culture. If this were so, restoration of the anti-hapten IgG response by TRF should also be achieved when hapten is coupled to a heterologous carrier protein. However, results with a change of carrier protein failed to support this expectation. Thus, DNP-CG, capable of inducing IgG anti-TNP PFC in cultures containing both T-

T cells in

an

325

IgG response to soluble antigens

Table 3. The effect of TRF removal on Day 1

DNP-KLH cells*

TRF (ml)

Antigent

0-5 05 0-5

0 + + + + +

10' Untreated Untreated 10' G ot T treated 107 G a T treated 107 G a T treated 107 G o T treated

Treatment of cultures on day 1

IgG PFC/culture on day 5 (s.e.)

32 12,912 925 10,965 10,631

(5 99) (1-87) (1-55) (1-02) (1-04) 10,106 (1-06)

-

-

Wash, replace TRF Wash, replace medium

* Donor mice primed and then boosted 7 days before killing. t 0-01 pg/ml DNP-KLH.

Table 4. TRF and challenge with heterologous carrier-hapten IgG PFC per 107 cultured (s.e.) DNP primed cells

Cells per culture 0 01 ug/ml DNP primed*

107 DNP-KLH 107 G a T DNP-KLH 107 G a T DNP-KLH 107 G a T DNP-KLH 107 G ct T DNP-KLH Sx 106 G a T DNP-KLH 107 DNP-CG 107 G a T, DNP-CG 107 G ae T, DNP-CG 107 G ae T, DNP-CG 7x 106 G or T, DNP-CG

Carrier primed* 5x 106 CG -

3x 106 KLH

Additions

antigen

Medium Medium TRF TRF TRF Medium Medium TRF TRF

DNP-KLH DNP-KLH DNP-KLH 0 DNP-CG DNP-CG DNP-CG 0 DNP-CG DNP-KLH DNP-KLH

TRF Medium

Expt 1

Expt 2

9300 (-) 1733 (1-12)

35,294 (1-19) 50 (2-96) 883 (1-86) 4640 (1-35) n.d. 18 (3 54) 3255 (1-19) 139 (4-30) 7712 (1-17)

14,980 (1-04) 14 (2-80) 6,518 (1-13) 365 (1-47) 23,884 (1-08)

n.d. = Not done. * Donor mice primed and then boosted 7 days before killing.

depleted DNP-KLH p/b cells and CG primed cells, only poorly immunogenic in cultures of goat anti-T treated DNP KLH p/b cells and TRF (Table 4). Similarly, DNP-KLH failed to stimulate an IgG response in T cell-depleted DNP-CG p/b cells in the presence of TRF. One reason for this deficiency could be a change in antigen handling; in the absence of the original carrier protein, B cells are no longer either recognizing this molecule or producing antibody against it. We therefore tested restoration of the anti-hapten response of DNP-CG p/b by TRF after altering the physical form of the DNP-KLH. Both IgM and IgG responses were somewhat enhanced when DNP-KLH had been taken up by macrophages, or was presented in insoluble form (i.e. was

attached to Sepharose beads (Figs 4 and 5). In contrast to Feldmann et al. (1974), the anti-hapten response to Sepharose-antigen remained TRF dependent. The greatest enhancement of IgG response resulted when, in an attempt to favour antigen antibody lattice formation on the surface of specific DNP-binding B cells, we added an excess of anti-KLH globulin a few hours after cellular interaction with antigen. Thus alteration in presentation of antigen or its binding to B cells enhanced the ability of TRF to restore IgG responses of a T-depleted cell population to a hapten on a heterologous carrier protein. However, we did not obtain complete restoration of IgG responses to their original levels (untreated DNP-primed and boosted cells).

J. R. North, J. T. Kemshead & B. A. Askonas

326

6000

D 4000 CL

2000

DNP- DNP- MACRKLH DNP-KLH CG

.:., .

.,.,.,.

DNP- DNP- MACR- a b c CG KLH DNP-KLH DNPKLH +

ANTI

DNP-CG DNP-KLH a b c a b c DNP-KLH-Seph DNP-KLH-Seph

KLH Ab

Figure 4. Carrier change in the presence of TRF. Cultures of T cell-depleted DNP-CG p/b cells were stimulated with various antigen preparations in the presence of TRF. Both IgM (hatched columns) and IgG (stippled columns) PFC per culture, assayed on day 5, are shown after subtraction of back ground PFC (cultures without antigen). Error bars represent the standard errors in the total PFC response of replicate cultures. DNP-CG: 0 01 jig/ml. DNP17CG; DNP-KLH: 0-01 fig/ml. DNP91KLH; MACR-DNP KLH: 5x 105 normal peritoneal cells pre-incubated with DNP KLH and washed before addition to culture; Anti-KLH Ab: anti-KLH globulins added to cultures 3 h after 0 01 ag/ml DNP91KLH to give a final anti KLH antibody concentration of (a) 0 3, (b) 0-16 and (c) 0-08 ig Ab/ml.

Antigen requirement during various phases of culture We have shown previously (North & Askonas, 1976) that the IgG response in vitro depends entirely upon addition of antigen to the cultures. Here, we questioned whether free antigen was required continuously. Since the majority of cell-bound antigen might be bound to macrophages and other adherent cells, we transferred non-adherent cells, after washing, from dishes containing antigen to other culture dishes containing the adherent cells from antigenfree cultures. Appropriate control transfers were also made. This attempt at antigen removal, markedly reduced the response when performed on day 1 (Fig. 6). Washing and transfer of non-adherent cells on subsequent days gave a rapidly lessening effect-suggesting either that cell-bound antigen becomes sufficient after day 2, or that B-cell proliferation and maturation becomes antigen independent during this

TRF:

+

+

+

-

Figure 5. Carrier change in the presence of TRF. 107 T celldepleted DNP-CG p/b cells were stimulated with DNPKLH-Sepharose in the presence or absence of TRF. Both IgM (hatched columns) and IgG (stippled columns) PFC per culture, assayed on day 5, are shown. Error bars represent the standard errors in the total PFC response of replicate cultures. DNP-KLH-Seph: sepharose 4B beads coupled with 200 /ig DNP91KLH per ml (wet volume) of gel. (a) Approximately 75,000 beads/culture; (b) approximately 25,000 beads/culture; (c) Approximately 2500 beads/culture.

period. Non adhering cells taken after 1-4 days of culture without antigen contact, mixed with antigen and antigen-bearing adherent cells, produced only feeble responses. DISCUSSION An IgG anti-hapten response in Mishell-Dutton cultures of primed and boosted spleen cells arises from a subpopulation of B cells previously activated in the donor animal (North & Askonas, 1976). Such intermediate B cells derive from a long lived memory cell population and have a lowered buoyant density suggesting recent division. Our in vitro studies therefore focus upon the later part of a maturation sequence though most IgG precursors continue to divide during the entire 5-day culture period (Kemshead et al., 1977). Antigen is still required in vitro for the appearance of IgG PFC, even though cells

327

T cells in an IgG response to soluble antigens

I

15,000 In

a) c

Q 0

10,000

0~

-.A.

'

(9)

~~~~~---A±

--

/

5000k

2

ULtreated

3

4

Day of treatment

Figure 6. Timing of antigen requirement. Cultures of DNP-KLH p/b cells were set up in the usual way with or without DNPKLH as antigen, and at the times indicated, non-adherent cells were removed and washed, leaving adherent cells in the dishes. Non-adherent cells were then returned to the dishes containing adherent cells in combinations listed below, with or without the addition of fresh antigen, and incubation was resumed until final harvest and plaque assay on day 5.

Previous antigen exposure of:

Non-adherent Adherent Fresh antigen after wash cells cells x A 0 A

Continuous antigen Antigen removal Delayed antigen addition No antigen

had been recently exposed to antigen in vivo, but the antigen requirement changes between days 1 and 2 of culture (Fig. 6). Whether cellular proliferation has become antigen-independent after day 2 of culture, or whether cell-bound antigen then becomes sufficient is not yet clear. Similarly, a primary response in vitro requires antigen for only 72 h (Pierce, 1973) and modification in antigen requirement is also suggested by studies in vivo. A marked change in susceptibility of the clonal antibody response to agents which interfere with antigen-cell interactions, such as DNP-S3 (Mitchell, Humphrey & Williamson, 1972) and anti-hapten antibody, was noted between days 2 and 4 after adoptive transfer (Askonas, McMichael & Roux, 1976). The IgG response of intact mice to DNP-KLH

+

+ +

+ -

-

-

+

+

-

-

is dependent on thymus-derived (T) cell recognition of the carrier KLH (Raff, 1970). This T dependence is true of the late phases of memory B-cell maturation to IgG secretion (Table 1) and also appears necessary for the generation of intermediate activated B cells. Spleen cells from DNP-KLH primed donors boosted with DNP-CG, cultured with additional carrierprimed cells, have failed to show IgG anti DNP responses. Our data (Table 2) show that a soluble, non antigen specific factor (TRF) produced in allogeneic spleen cell cultures can restore the IgG response of primed, T deprived cultures to the soluble antigen DNP-KLH. Restoration of IgM responses to foreign erythrocytes in T cell-deprived cultures by similar allogeneic culture supernatants have been reported by many authors (Britton, 1972; Schimpl & Wecker,

328

J. R. North, J. T. Kemshead & B. A. Askonas

1972a; Watson, 1973; Waldmann, 1975; Dutton (review), 1975). Restoration of responses to soluble antigens has proved difficult (Feldmann & Basten, 1972; Waldmann, 1974; Henry, 1975; Armerding & Katz, 1974). The latter authors showed that allogeneic supernatants were only capable of magnifying IgM and IgG responses to DNP-KLH and DNP-CG in response to occasional antigen preparations or when soluble antigen was carried by macrophages. The most marked difference in procedure between our experiments (Table 2) and those of Waldmann (1975) and Armerding & Katz (1974) is that we have not changed the carrier antigen but have depleted cultures of T cells. When, in addition to T-cell depletion, the carrier protein is changed (Table 4) we find that TRF is unable to restore the IgG response to soluble antigen. According to Schimpl & Wecker (1972) allogeneic culture supernatants also contain components which can expand residual carrierprimed T cells, but this activity is also present in supernatants of syngeneic cultures, whereas we do not detect any restorative capacity in such medium (Table 2). In view of the virtually complete ablation (98-99%) of the response in some experiments after T-cell depletion, the identical PFC kinetics and the absence of an effect of control supernatants it seems unlikely that TRF acts in our system as a T-cell expanding factor. The deficit in restorative capacity of TRF in cultures with heterologous carrier antigen can, to some extent, be reversed by modifying the physical form of the antigen or its interaction with cell receptors. Thus DNP-KLH presented bound to macrophages (Fig. 4), or on the surface of Sepharose beads (Fig. 5) can stimulate an anti-DNP response in T-depleted cultures of DNP-CG p/b cells with TRF. Particulate presentation of antigen might encourage multiple B cell-antigen interactions, which may be essential for B-cell triggering. However, the restoration of response by TRF in T-depleted cultures stimulated with homologous soluble antigen (Table 2) does not fit this hypothesis. Interpretation is complicated by the presence of anti-carrier-specific B cells in culture which could bind soluble antigen to their surface receptors, forming hapten-coated particles. Furthermore, some anti-carrier antibody will be secreted early in culture and encourage lattice formation. Enhancement of the IgG response when we add anti-carrier antibody to T-depleted cells incubated with a heterologous carrier-hapten conjugate and TRF, (Fig. 4) favours this possibility. Similar

speculations have been made after observing that anti-carrier antibody can augment anti-hapten responses in vivo (McBride & Schierman, 1971; Janeway, Koren & Paul, 1975), and that antigenspecific suicide of carrier-binding B cells reduces anti-hapten responses in vitro and in vivo (Kirov & Parish, 1976). All data appear consistent with the generalization that a non-specific T-cell factor can replace specific T-cell function in vitro in responses to particulate antigens, cell-bound antigens and soluble antigens complexed with antibody, but not to soluble antigens alone. This factor is required during the last few days of proliferation before final maturation into IgG secretion commences. It must be added to cultures of T-depleted cells at the time of initiation (Fig. 2) as only 20-25% of maximal restoration is achieved if addition is delayed 24 h. This coincides with the observation of Schimpl & Wecker (1973) that although TRF addition on day 2 is optimal for IgM responses, the factor must be added at time zero for restoration of IgG responses to SRBC. Our data differ in that we find that a single TRF addition gives a dose dependent restoration of IgG response (Fig. 1)-occasionally greater than the untreated response (Table 2). The presence of TRF is required only for the first 24 h (Table 3), indicating either that a single non-specific T cell-derived triggering event is sufficient or that the factor remains available bound to cells in culture since TRF is absorbed by activated cells (Schimpl & Wecker, 1972b). Earlier conclusions from studying the restoration of an IgM anti-SRBC response by TRF indicate that the factors are only necessary to trigger final maturation and that PFC precursors are dividing before and independently of TRF addition (Dutton, 1975; Askonas, Schimpl & Wecker, 1974). The present results imply either that TRF is required for the initiation of division of pre-activated B cells when they are placed in culture or alternatively, and more consistent with the view discussed above (Askonas et al., 1974; Dutton, 1975), that B-cell maturation into IgG secretion has to be initiated by TRF during proliferation and is followed by several further proliferative cycles before we see IgG secretion. We have no evidence to distinguish between these alternative models.

REFERENCES AMERDING D. & KATZ D.H. (1974) Biological and bio-

T cells in an IgG response to soluble antigens chemical properties of an allogeneic effect factor (AEF) active in triggering specific B lymphocytes. J. exp. Med. 140, 19. ASKONAs B.A., SCHIMPL A. & WECKER E. (1974) The differentiation function of T cell replacing factor in nu/nu spleen cell cultures. Europ. J. Immunol. 4, 164. ASKONAs B.A., MCMICHAEL A.J. & Roux M.E. (1976) Clonal dominance and the preservation of clonal memory cells mediated by antigen-antibody. Immunology, 31, 541. BRETSCHER P.A. (1975) The Two signal model for B cell induction. Transplant. Rev. 23, 37. BRI-rON S. (1972) When allogeneic mouse spleen cells are mixed in vitro the T cells secrete a product which guides the maturation of B cells. Scand. J. Immunol. 1, 89. DRESSER D.W. & GREAVES M.F. (1973) Assays for antibody producing cells in Handbook of Experimental Immunology, (ed. by D.M. Weir), Blackwell Scientific Publications, Oxford. DUTTON R.W. (1975) Separate signals for the initiation of proliferation and differentiation in the B cell response to antigen. Transplant. Rev. 23, 66. FELDMANN M. & BASTEN A. (1972) Cell interactions in the immune response in vitro. IV Comparison of the effects of antigen-specific and allogeneic thymus derived cell factors. J. exp. Med. 136, 722. FELDMANN M., GREAVES M.F., PARKER D.C. & RITTENBERG M.B. (1974) Direct triggering of B lymphocytes by insolubilised antigen. Europ. J. Immunol. 4, 591. GOLUB E.S. (1971) Brain associated 0 antigen: Reactivity of rabbit anti mouse brain with mouse lymphoid cells. Cell. Immunol. 2, 353. HENRY C. (1975) In vitro anti hapten response to a lactoside conjugated protein. Cell. Immunol. 19, 117. JANEWAY C.A., KOREN H.S. & PAUL W.E. (1975) The role of thymus derived lymphocytes in an antibody mediated hapten-specific helper effect. Europ. J. Immunol. 5, 17. KEMSHEAD J.T., NORTH J.R. & ASKONAS B.A. (1977) IgG anti-hapten antibody secretion in vitro commences after extensive precursor proliferation. Immunology (In Press). KIROV S.M. & PARISH C.R. (1976) Evidence that carrier specific B cells play a role in cellular collaboration. Scand. J. Immunol. 5, 191. McBRIDE R.A. & SCHIERMAN L.W. (1971) Passive antibody

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and the immune response. Factors which determine enhancement and suppression. J. exp. Med. 134, 833. MISHELL R.I. & DUTTON R.W. (1967) Immunisation of dissociated spleen cell cultures from normal mice. J. exp. Med. 126, 423. MITCHELL G.F., HUMPHREY J.H. & WILLIAMSON A.R. (1972) Inhibition of secondary anti-hapten responses with the hapten conjugated to type 3 Pneumococcal Polysaccharide. Europ. J. Immunol. 21, 460. MOLLER G. (1975) One non specific signal triggers B lymphocytes. Transplant. Rev. 23, 126. NORTH J.R. & ASKONAs B.A. (1976) IgG response in vitro. I The requirement for an intermediate responsive cell type. Europ. J. Immunol. 6, 8. PARISH C.R., KIROV, S.M., BOWERN N. & BLANDEN N. & BLANDEN R.V. (1974) A one step procedure for separating mouse T and B lymphocytes. Europ. J. Immunol. 4, 808. PIERCE C.W. (1973) Immune responses in vitro. VI. Cell interactions in the development of primary IgM, IgG and IgA plaque forming cell responses in vitro. Cell. Immunol. 9, 453. RAFF M.C. (1970) Role of thymus derived lymphocytes in the secondary humoral immune response in mice. Nature (Lond.), 226, 1257. RITTENBERG M.B. & PRATT K.L. (1969) Antitrinitrophenyl (TNP) Plaque assay. Primary response of mice to soluble and particulate immunogen. Proc. Soc. exp. Biol. (N. Y.), 132, 575. SCHIMPL A. & WECKER E. (1972a) Replacement of T cell function by a T cell product. Nature: New Biology, 237, 15. SCHIMPL A. & WECKER E. (1972b) Studies on the source and action of the T cell replacing factor (TRF). Advanc. exp. med. Biol. 29, 179. SCHIMPL A. & WECKER E. (1973) Stimulation of IgG antibody response in vitro by T cell replacing factor. J. exp. Med. 137, 547. WALDMANN H. (1975) T cell dependent mediator in the immune response. III. The role of non-specific factor (NSF) in the in vitro immune response. Immunology, 28, 497. WATSON J. (1973) The role of humoral factors in the initiation of in vitro primary immune responses. J. Immunol. 111, 1301.