Immunology, 1975, 28, 1149.
T Cell-dependent Mediator and B-cell Clones I. LEFKOVITS,* J. QUINTANS,* A. MUNROt AND H. WALDMANNt * Basel Institute for Immunology, Basel, Switzerland, and t Immunology Division, Department of Pathology, University of Cambridge, Cambridge
(Received 14th October 1974; acceptedfor publication 15th November 1974) Summary. Supernatants were collected from keyhole limpet haemocyanin (KLH) primed spleen cells which had been incubated in tissue culture medium with their priming antigen KLH. These non-specific factor (NSF) containing supernatants were then tested in a microculture system for their ability to facilitate an antiSRBC response of nu/nu or AT x BM spleen cells. It was concluded that: (a) NSF was able to engage a large number ofthe available pool ofsheep erythrocyte (SRBC) specific B cells into an antibody response; (b) this response was characterized by the development of clones expressing plaque-forming cells (PFC), the number of PFC produced within a clone being dependent upon the amount of NSF available in that culture; (c) NSF probably acted directly on B cells, and not via other accessory cell types. INTRODUCTION In the previous paper we have shown that keyhole limpet haemocyanin (KLH) primed spleen cells are capable of engaging a large number of B cells for an antibody response against the two particulate antigens sheep erythrocytes (SRBC) and donkey erythrocytes (DRBC). It has been previously shown that a non-specific factor (NSF) could be elicited from spleen cell cultures containing KLH-activated T cells which was also able to facilitate the response of B cells to such particulate antigens (Waldmann and Munro, 1973). We here show that NSF is probably able to exert its facilitatory capacity by acting directly on B cells responding to their challenge antigen, and not via other accessory cells. This activity would appear to involve the participation of available antigen-specific B-cell precursors in antibody production, and also to determine the extent to which these precursors could develop into detectable clones of antibody-forming cells. MATERIALS AND METHODS Mice, antigens, culture conditions and assay methods These were similar to those described in the previous paper. In some experiments nude C57B1/6 mice were used (Bomholtgaard, By, Denmark). Production of active supernatants (SUP (KLH)) Spleen cells from KLH-primed CBA/Ca mice were incubated with KLH (10 Yg/ml) for 48 hours in tissue culture medium. After 48 hours the cells were harvested, and separated Correspondence: Dr H. Waldmann, Immunology Division, Department of Pathology, Tennis Court Road,
Cambridge.
1149
L Lefkovits et al. 1150 from the supernatant by centrifugation. This supernatant material was stored in small aliquots in liquid nitrogen until use. In the text we refer to the 'supernatant' as SUP (KLH) and to its presumed active moiety as NSF. RESULTS (1) THE EFFECT OF SUP (KLH) ON THE NUMBER OF PRECURSOR B CELLS INITIATED INTO AN ANTIBODY RESPONSE
This first experiment was concerned with the problem of whether a supernatant produced from activated T cells (SUP (KLH)) would mimic the effect of those same cells in involving antigen-specific B cells in antibody production. The results presented in Table 1 TABLE 1 HELPER
ACTIVITY EXPRESSED BY
'SUPERNATANTS'
Fraction of nonresponding
Source of B cells CBA spleen cells from AT x BM mice* 105
BALB/c nude spleen cellst
with supernatant
Nil Nil
10'
105
10s
Supplementation
105
10'
SUP SUP SUP SUP
(KLH)§ (KLH) (KLH) (KLH)
microculturest
SRBC (50 pl per ml) (1 per cent)
F0
+ + + + -
0-98 1 0*61 0-33 1 1
95 per cent confidence limits
094-0*99 0-98-1 0-53-0-68 0-27-0 41
0-98-1 098-1
* AT x BM mice: adult thymectomized, irradiated and bone marrow-protected mice. t From fourth backcross. I Each tabular entry based on spot tests from 180 microcultures. § SUP (KLH) = supernatant from KLH-primed spleen cells cultured in the presence of KLH.
confirm this contention and furthermore strongly suggest that the effect of the supernatant is not exerted through SRBC-specific T cells, as these would be extremely limiting in the microculture wells. SUP (KLH) ON THE NUMBER OF PFC EXPRESSED BY EACH B-CELL PRECURSOR RESPONDING TO SRBC In general terms the microculture system allows us to estimate both the number of precursor B cells that are involved in a response, and the size of that part of the clonal progeny which can express as PFC. In the experiments to be described we analyse the second of these parameters. A small number of nude spleen cells was cultured in the presence of SRBC and various test supplements. On days 3-5 of the experiment individual wells were assayed for their PFC content. Fig. 1 and Table 2 present the relevant data. It can be seen that in the absence of any further supplementation virtually no microcultures (of sixty established) responded to SRBC. In the presence of 5 pl of SUP (KLH) per well a peak of responding wells was obtained on days 4 and 5 and in fact half of the sixty microcultures produced a
(2)
THE EFFECT OF
1151
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1152 THE
EFFECT OF
al.
TABLE 2 'NSF-CONTAINING SUPERNATANT
ON
2 x 104 nude spleen cells and 1-6 x 105 irradiated nude
Expt no.
spleen cells per wellt supplemented with 1 2 3
Lefkovits
et
I.
B-CELL PRECURSOR ACTIVATION*
Day 3
Day 5
E§
MdT
F0
Z
MdT
F0
c
Md
n.d. 4-6 3
n.d. 5 3
0 88 05 0 58
n.d. 29 16
n.d. 29 14
0 93 05 0 25
n.d. 46 41-5
n.d. 44 57
F0o
Nil 095 0 78 SUP (KLH)** 2 x 104 irradiated allogeneic cells 0 78
Day 4
n.d. = Not determined because there were too few responding wells. * Based on microcultures containing two or more PFC (for each treatment sixty wells were assayed). t SRBC were added to all cultures. I F0 = fraction of non-responding microcultures. § c = Average clone size (calculated as previously described Quintans and Lefkovits, 1974). ¶ Md = median number of PFC/well. ** SUP (KLH) stands for 'NSF-containing supernatant' (5 pl/well).
response. A positive control for this experiment was achieved with the addition of 2 x 1O' irradiated allogeneic spleen cells where by day 5 only 27 per cent of the cultures were nonresponding. This experiment shows that a substantial proportion of the available cell precursors could be affected at this dilution of SUP (KLH). The estimation of average clone size indicates that 5 1A of SUP (KLH) per well is effective in promoting detectable clonal expansion and by day 5 of the culture period each precursor cell was estimated to be producing an average of forty-six PFC (line 2). This number is comparable to that achieved with allogeneic stimulation (line 3). In order to establish that NSF was directly influencing clonal events rather than solely (if at all) initiation events we performed a titration of SUP (KLH) and calculated the 4-day clone size for each dilution. The data in Fig. 2 and Table 3 suggest that as the amount of SUP (KLH) in each culture is reduced, the number of responding wells, and the average estimated clone size, diminish. This indicates that NSF is indeed involved in the expression of clonal potential in terms of PFC arising from a single precursor. TABLE 3 THE EFFECT OF NSF-CONTAINING SUPERNATANT ON THE SIZE OF CLONES PRECURSOR B CELL
2 x 104 nude spleen cells + 1 6 x 10 irradiated nude spleen cells per well* supplemented with: SUP (KLH)§ (5 pl/well) SUP (KLH) (2-5 Al/well) SUP (KLH) (1-25 pl/well) Nil
Fraction of
PFC per tray:
non-responding culturest 0.1 0-18 03 0 97
3972 2266 1283 9
OF
PFC
DERIVED FROM ONE
Estimated number of clones per tray:
138 101-4 72 2
Estimated average clone size 28 8
22-3 17-8 n.d.
n.d. = Not determined. *
SRBO were added to all cultures.
t Based on the PFC analysis on day 4. Less than two PFC per well constituted a negative response. 1 tray = sixty microcultures. § SUP (KLH) stands for 'NSF-containing supernatant'.
1153
T Cell-dependent Mediator 60 -(a)
SIf
50
40
0
30
4
20 10
50~~~~~~~~~~~~~~~~~~~~~~~~~95
-aa) am
6040 ( b), 30 30 _
§l"
---
a)
0
E z
60- (c)
O
L
P
-
20
10_
-1
H
2
4
6
K)
20 4060 O00 PFC per well
200 400
FIG. 2. Titration of SUP (KLH) and clonal expression. Microcultures containing 2 x 104 C57BI/6 nude spleen cells and 1-7 x 105 irradiated nude spleen cells were challenged with SRBC in the presence of (a) 5 jul, (b) 2-5 pl and (c) 1-25 4u1 of SUP (KLH) per well. After 4 days in culture PFC responses to individual microculture wells were determined, and a cumulative plot of the results produced as in Fig. 1. FO = fraction of non-responding cultures. F+ = fraction of responding cultures.
DISCUSSION In our preceding paper (Waldmann, Lefkovits and Quintains, 1975) we attempted to probe the mechanism by which 'helper' T cells could manifest 'non-specific co-operation'. We determined that these cells could influence the number of B-cell precursors participating in the antibody response. The use of supernatants from such activated cells can allow us to focus our attention on the behaviour of responding B cells, inasmuch as each well receives a constant dose of the active moiety NSF. This eliminates from our analysis the consequences of heterogeneity of T cells which might have been added to each well. With this in mind, these experiments were initiated to seek an answer to what now appears a rather naive question. This was: 'Does NSF act by initiating precursors into antibody response, or does it act by expanding the clone size ofinitiated precursors?' It has emerged from our studies that the effect of this factor is apparent on both these components. However, our detection system only permits us to score a positive response where clonal proliferation is expressed in the form of antibody-secreting cells. Any stimulus which established a hidden pool of proliferating cells would escape detection. It is also clear that any agent which alters the number of PFC expressed by clones is likely (by definition) to affect the number of responding precursor cells we detect. We can only say from our
1154 I. Lefkovits et al. results that NSF can influence the number of PFC a clone expresses, and cannot argue that it serves only as a clone initiator or a clone expander. Analysis of our cumulative plots make it clear that NSF drives B cells to a similar distribution of responses as achieved by another conventional stimulus (allogeneic cells). It should be noted that the interquartile ranges we observed were barely dissimilar to the positive control groups. Therefore we feel that NSF was not selecting a privileged set ofB-cell precursors, but was capable ofengaging the majority of the available pool. Furthermore, it seems likely that NSF exerted its action directly upon B cells and not through SRBC-specific T cells, in as far as we assayed its activity on very small numbers of nude or AT x BM spleen cells where T cells should have been extremely limiting. This argument stands in contrast to that of Gorczynski, Miller and Phillips (1973) who implied that 'allogeneic factor' needed to act via SRBC-specific T cells in order to exert a stimulatory effect on B cells. It also seems unlikely to us that NSF needs to act via macrophages because our preliminary (unpublished) studies show that NSF can restore the capacity of macrophage-deficient thoracic duct lymphocytes to make anti-SRBC responses in the presence of 2-mercaptoethanol. Hunter and Kettman (1974) have recently reported on the activity of an allogeneic factor in antibody responses to TNP and SRBC. Although they have not titrated the activity of the supernatant, their results are in general agreement with those presented in this paper. Hunter and Kettman (1974) suggested that an allogeneic factor can expand clones already 'initiated' by the T cell-independent antigen TNP-LPS (TNP-lipopolysaccharide). Our studies with TNP-LPS (unpublished results) suggest that both allogeneic factor and NSF can produce an increase in the detectable background response to TNP. This obscures any evaluation of the activity of these factors on the behaviour of clones already initiated by thymus-independent antigens. It remains to be seen whether allogeneic factors and NSF are identical. Much evidence is now accumulating that B cells are capable of mounting a proliferative response to SRBC in the absence of T cells or their factors (Askonas, Schimpl and Wecker, 1974; Dutton, 1974) and that T-cell factors of the 'non-specific' type act merely as differentiation mediators on this hidden proliferating pool. Our data are certainly not incompatible with this possibility. ACKNOWLEDGMENTS We gratefully acknowledge the skilful technical assistance of Anita Soderberg and Susan Davies. H.W. was supported by grants from the Medical Research Council. REFERENCES
ASKONAS, B., SCHIMPL, A. and WECKER, E. (1974). 'The differentiation function of T cell replacing factor in nu/nu spleen cell cultures.' Europ. J.
Immunol., 4, 164. DUTTON, R. W. (1974). 'T cell factors in the regulation of the B cell response.' The Immune System. Genes, Receptors and Signals (ed. by E. C. Sercarz and A. R. Williamson, p. 485. Academic Press, New York. GORCZYNSKI, R. M., MILLER, R. G. and PHILLIPs, R. A. (1973). 'Reconstitution of T cell-depleted spleen cell populations by factors derived from T cell. III. Mechanism of action of T cell-derived factors.' J. Immunol., 111, 900. HUNTER, P. and KETTMAN, J. R. (1974). 'Mode of
action of a supernatant activity from T-cell cultures that nonspecifically stimulates the immune response.' Proc. nat. Acad. Sci. (Wash.), 71, 512. QUINTANS, J. and LEFKOVITs, I. (1974). 'Clones of antibody-forming cells in pokeweed mitogen stimulated microcultures. II. Estimation of the frequency of precursor cells and the average clone size.' Europ. J. Immunol., 4, 617. WALDMANN, H. and MUNRO, A. J. (1974). 'T cell dependent mediator in the immune response. II. Physical and biological properties.' Immunology, 27,53. WALDMANN, H., LEFKOVITS, I. and QUINTANSJ. (1975). 'Limiting dilution analysis of helper T-cell function.' Immunology, 28, 1135.
T Cell-dependent Mediator and B-cell Clones I. LEFKOVITS,* J. QUINTANS,* A. MUNROt AND H. WALDMANNt * Basel Institute for Immunology, Basel, Switzerland, and t Immunology Division, Department of Pathology, University of Cambridge, Cambridge
(Received 14th October 1974; acceptedfor publication 15th November 1974) Summary. Supernatants were collected from keyhole limpet haemocyanin (KLH) primed spleen cells which had been incubated in tissue culture medium with their priming antigen KLH. These non-specific factor (NSF) containing supernatants were then tested in a microculture system for their ability to facilitate an antiSRBC response of nu/nu or AT x BM spleen cells. It was concluded that: (a) NSF was able to engage a large number ofthe available pool ofsheep erythrocyte (SRBC) specific B cells into an antibody response; (b) this response was characterized by the development of clones expressing plaque-forming cells (PFC), the number of PFC produced within a clone being dependent upon the amount of NSF available in that culture; (c) NSF probably acted directly on B cells, and not via other accessory cell types. INTRODUCTION In the previous paper we have shown that keyhole limpet haemocyanin (KLH) primed spleen cells are capable of engaging a large number of B cells for an antibody response against the two particulate antigens sheep erythrocytes (SRBC) and donkey erythrocytes (DRBC). It has been previously shown that a non-specific factor (NSF) could be elicited from spleen cell cultures containing KLH-activated T cells which was also able to facilitate the response of B cells to such particulate antigens (Waldmann and Munro, 1973). We here show that NSF is probably able to exert its facilitatory capacity by acting directly on B cells responding to their challenge antigen, and not via other accessory cells. This activity would appear to involve the participation of available antigen-specific B-cell precursors in antibody production, and also to determine the extent to which these precursors could develop into detectable clones of antibody-forming cells. MATERIALS AND METHODS Mice, antigens, culture conditions and assay methods These were similar to those described in the previous paper. In some experiments nude C57B1/6 mice were used (Bomholtgaard, By, Denmark). Production of active supernatants (SUP (KLH)) Spleen cells from KLH-primed CBA/Ca mice were incubated with KLH (10 Yg/ml) for 48 hours in tissue culture medium. After 48 hours the cells were harvested, and separated Correspondence: Dr H. Waldmann, Immunology Division, Department of Pathology, Tennis Court Road,
Cambridge.
1149
L Lefkovits et al. 1150 from the supernatant by centrifugation. This supernatant material was stored in small aliquots in liquid nitrogen until use. In the text we refer to the 'supernatant' as SUP (KLH) and to its presumed active moiety as NSF. RESULTS (1) THE EFFECT OF SUP (KLH) ON THE NUMBER OF PRECURSOR B CELLS INITIATED INTO AN ANTIBODY RESPONSE
This first experiment was concerned with the problem of whether a supernatant produced from activated T cells (SUP (KLH)) would mimic the effect of those same cells in involving antigen-specific B cells in antibody production. The results presented in Table 1 TABLE 1 HELPER
ACTIVITY EXPRESSED BY
'SUPERNATANTS'
Fraction of nonresponding
Source of B cells CBA spleen cells from AT x BM mice* 105
BALB/c nude spleen cellst
with supernatant
Nil Nil
10'
105
10s
Supplementation
105
10'
SUP SUP SUP SUP
(KLH)§ (KLH) (KLH) (KLH)
microculturest
SRBC (50 pl per ml) (1 per cent)
F0
+ + + + -
0-98 1 0*61 0-33 1 1
95 per cent confidence limits
094-0*99 0-98-1 0-53-0-68 0-27-0 41
0-98-1 098-1
* AT x BM mice: adult thymectomized, irradiated and bone marrow-protected mice. t From fourth backcross. I Each tabular entry based on spot tests from 180 microcultures. § SUP (KLH) = supernatant from KLH-primed spleen cells cultured in the presence of KLH.
confirm this contention and furthermore strongly suggest that the effect of the supernatant is not exerted through SRBC-specific T cells, as these would be extremely limiting in the microculture wells. SUP (KLH) ON THE NUMBER OF PFC EXPRESSED BY EACH B-CELL PRECURSOR RESPONDING TO SRBC In general terms the microculture system allows us to estimate both the number of precursor B cells that are involved in a response, and the size of that part of the clonal progeny which can express as PFC. In the experiments to be described we analyse the second of these parameters. A small number of nude spleen cells was cultured in the presence of SRBC and various test supplements. On days 3-5 of the experiment individual wells were assayed for their PFC content. Fig. 1 and Table 2 present the relevant data. It can be seen that in the absence of any further supplementation virtually no microcultures (of sixty established) responded to SRBC. In the presence of 5 pl of SUP (KLH) per well a peak of responding wells was obtained on days 4 and 5 and in fact half of the sixty microcultures produced a
(2)
THE EFFECT OF
1151
T Cell-dependent Mediator "-:' ' ':8L;0OTT." """
, ....*
Zb,.= *.
=
I_ _I
. -..
1
I_.::.0
*
1
111
-i I_
'
_
I
-
L
I
'......
4 ' °" '' '''
4.0
U.
a.
D
,
crfiiC~~~~~~~...
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.
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1,
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r4)
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1152 THE
EFFECT OF
al.
TABLE 2 'NSF-CONTAINING SUPERNATANT
ON
2 x 104 nude spleen cells and 1-6 x 105 irradiated nude
Expt no.
spleen cells per wellt supplemented with 1 2 3
Lefkovits
et
I.
B-CELL PRECURSOR ACTIVATION*
Day 3
Day 5
E§
MdT
F0
Z
MdT
F0
c
Md
n.d. 4-6 3
n.d. 5 3
0 88 05 0 58
n.d. 29 16
n.d. 29 14
0 93 05 0 25
n.d. 46 41-5
n.d. 44 57
F0o
Nil 095 0 78 SUP (KLH)** 2 x 104 irradiated allogeneic cells 0 78
Day 4
n.d. = Not determined because there were too few responding wells. * Based on microcultures containing two or more PFC (for each treatment sixty wells were assayed). t SRBC were added to all cultures. I F0 = fraction of non-responding microcultures. § c = Average clone size (calculated as previously described Quintans and Lefkovits, 1974). ¶ Md = median number of PFC/well. ** SUP (KLH) stands for 'NSF-containing supernatant' (5 pl/well).
response. A positive control for this experiment was achieved with the addition of 2 x 1O' irradiated allogeneic spleen cells where by day 5 only 27 per cent of the cultures were nonresponding. This experiment shows that a substantial proportion of the available cell precursors could be affected at this dilution of SUP (KLH). The estimation of average clone size indicates that 5 1A of SUP (KLH) per well is effective in promoting detectable clonal expansion and by day 5 of the culture period each precursor cell was estimated to be producing an average of forty-six PFC (line 2). This number is comparable to that achieved with allogeneic stimulation (line 3). In order to establish that NSF was directly influencing clonal events rather than solely (if at all) initiation events we performed a titration of SUP (KLH) and calculated the 4-day clone size for each dilution. The data in Fig. 2 and Table 3 suggest that as the amount of SUP (KLH) in each culture is reduced, the number of responding wells, and the average estimated clone size, diminish. This indicates that NSF is indeed involved in the expression of clonal potential in terms of PFC arising from a single precursor. TABLE 3 THE EFFECT OF NSF-CONTAINING SUPERNATANT ON THE SIZE OF CLONES PRECURSOR B CELL
2 x 104 nude spleen cells + 1 6 x 10 irradiated nude spleen cells per well* supplemented with: SUP (KLH)§ (5 pl/well) SUP (KLH) (2-5 Al/well) SUP (KLH) (1-25 pl/well) Nil
Fraction of
PFC per tray:
non-responding culturest 0.1 0-18 03 0 97
3972 2266 1283 9
OF
PFC
DERIVED FROM ONE
Estimated number of clones per tray:
138 101-4 72 2
Estimated average clone size 28 8
22-3 17-8 n.d.
n.d. = Not determined. *
SRBO were added to all cultures.
t Based on the PFC analysis on day 4. Less than two PFC per well constituted a negative response. 1 tray = sixty microcultures. § SUP (KLH) stands for 'NSF-containing supernatant'.
1153
T Cell-dependent Mediator 60 -(a)
SIf
50
40
0
30
4
20 10
50~~~~~~~~~~~~~~~~~~~~~~~~~95
-aa) am
6040 ( b), 30 30 _
§l"
---
a)
0
E z
60- (c)
O
L
P
-
20
10_
-1
H
2
4
6
K)
20 4060 O00 PFC per well
200 400
FIG. 2. Titration of SUP (KLH) and clonal expression. Microcultures containing 2 x 104 C57BI/6 nude spleen cells and 1-7 x 105 irradiated nude spleen cells were challenged with SRBC in the presence of (a) 5 jul, (b) 2-5 pl and (c) 1-25 4u1 of SUP (KLH) per well. After 4 days in culture PFC responses to individual microculture wells were determined, and a cumulative plot of the results produced as in Fig. 1. FO = fraction of non-responding cultures. F+ = fraction of responding cultures.
DISCUSSION In our preceding paper (Waldmann, Lefkovits and Quintains, 1975) we attempted to probe the mechanism by which 'helper' T cells could manifest 'non-specific co-operation'. We determined that these cells could influence the number of B-cell precursors participating in the antibody response. The use of supernatants from such activated cells can allow us to focus our attention on the behaviour of responding B cells, inasmuch as each well receives a constant dose of the active moiety NSF. This eliminates from our analysis the consequences of heterogeneity of T cells which might have been added to each well. With this in mind, these experiments were initiated to seek an answer to what now appears a rather naive question. This was: 'Does NSF act by initiating precursors into antibody response, or does it act by expanding the clone size ofinitiated precursors?' It has emerged from our studies that the effect of this factor is apparent on both these components. However, our detection system only permits us to score a positive response where clonal proliferation is expressed in the form of antibody-secreting cells. Any stimulus which established a hidden pool of proliferating cells would escape detection. It is also clear that any agent which alters the number of PFC expressed by clones is likely (by definition) to affect the number of responding precursor cells we detect. We can only say from our
1154 I. Lefkovits et al. results that NSF can influence the number of PFC a clone expresses, and cannot argue that it serves only as a clone initiator or a clone expander. Analysis of our cumulative plots make it clear that NSF drives B cells to a similar distribution of responses as achieved by another conventional stimulus (allogeneic cells). It should be noted that the interquartile ranges we observed were barely dissimilar to the positive control groups. Therefore we feel that NSF was not selecting a privileged set ofB-cell precursors, but was capable ofengaging the majority of the available pool. Furthermore, it seems likely that NSF exerted its action directly upon B cells and not through SRBC-specific T cells, in as far as we assayed its activity on very small numbers of nude or AT x BM spleen cells where T cells should have been extremely limiting. This argument stands in contrast to that of Gorczynski, Miller and Phillips (1973) who implied that 'allogeneic factor' needed to act via SRBC-specific T cells in order to exert a stimulatory effect on B cells. It also seems unlikely to us that NSF needs to act via macrophages because our preliminary (unpublished) studies show that NSF can restore the capacity of macrophage-deficient thoracic duct lymphocytes to make anti-SRBC responses in the presence of 2-mercaptoethanol. Hunter and Kettman (1974) have recently reported on the activity of an allogeneic factor in antibody responses to TNP and SRBC. Although they have not titrated the activity of the supernatant, their results are in general agreement with those presented in this paper. Hunter and Kettman (1974) suggested that an allogeneic factor can expand clones already 'initiated' by the T cell-independent antigen TNP-LPS (TNP-lipopolysaccharide). Our studies with TNP-LPS (unpublished results) suggest that both allogeneic factor and NSF can produce an increase in the detectable background response to TNP. This obscures any evaluation of the activity of these factors on the behaviour of clones already initiated by thymus-independent antigens. It remains to be seen whether allogeneic factors and NSF are identical. Much evidence is now accumulating that B cells are capable of mounting a proliferative response to SRBC in the absence of T cells or their factors (Askonas, Schimpl and Wecker, 1974; Dutton, 1974) and that T-cell factors of the 'non-specific' type act merely as differentiation mediators on this hidden proliferating pool. Our data are certainly not incompatible with this possibility. ACKNOWLEDGMENTS We gratefully acknowledge the skilful technical assistance of Anita Soderberg and Susan Davies. H.W. was supported by grants from the Medical Research Council. REFERENCES
ASKONAS, B., SCHIMPL, A. and WECKER, E. (1974). 'The differentiation function of T cell replacing factor in nu/nu spleen cell cultures.' Europ. J.
Immunol., 4, 164. DUTTON, R. W. (1974). 'T cell factors in the regulation of the B cell response.' The Immune System. Genes, Receptors and Signals (ed. by E. C. Sercarz and A. R. Williamson, p. 485. Academic Press, New York. GORCZYNSKI, R. M., MILLER, R. G. and PHILLIPs, R. A. (1973). 'Reconstitution of T cell-depleted spleen cell populations by factors derived from T cell. III. Mechanism of action of T cell-derived factors.' J. Immunol., 111, 900. HUNTER, P. and KETTMAN, J. R. (1974). 'Mode of
action of a supernatant activity from T-cell cultures that nonspecifically stimulates the immune response.' Proc. nat. Acad. Sci. (Wash.), 71, 512. QUINTANS, J. and LEFKOVITs, I. (1974). 'Clones of antibody-forming cells in pokeweed mitogen stimulated microcultures. II. Estimation of the frequency of precursor cells and the average clone size.' Europ. J. Immunol., 4, 617. WALDMANN, H. and MUNRO, A. J. (1974). 'T cell dependent mediator in the immune response. II. Physical and biological properties.' Immunology, 27,53. WALDMANN, H., LEFKOVITS, I. and QUINTANSJ. (1975). 'Limiting dilution analysis of helper T-cell function.' Immunology, 28, 1135.
