International Immunology, Vol 2, No 6
© 1990 Oxford University Press 0953-8178/90 $3.00
Persistence of memory B cells in mice deprived of T cell help Paulo Vieira1 and Klaus Rajewsky Institute for Genetics, University of Cologne, FRG 'Present address. DNAX, Palo Alto, CA 94304-1104, USA Key words: memory B cells, B cell memory, T cell help
The influence of T cell help on the Induction, maintenance, and recall of B cell memory to a T cell-dependent antigen was studied In mice. The antigen was a hapten coupled to a protein carrier, and helper T cells were eliminated before, during, or after immunization by treatment of the animals with antl-CD4 antibodies. B cell memory was quantified in an adoptive cell transfer system, transferring B cells from the primed animals together with carrier-primed syngeneic T cells. Antl-CD4 treatment completely inhibited the induction of primary and secondary responses and of B cell memory. In contrast, it did not affect established B cell memory over a period of at least 6 weeks. Consequently, this suggests that within this time range B cell memory to a T celldependent antigen does not rely on the continuous recruitment of naive B cells Into the memory compartment, and memory B cells need little or no (antigen-mediated) T cell help In order to persist. Introduction Memory B cells are usually defined as cells which make secondary responses to thymus-dependent antigens (1,2). They easily giveriseto antibody responses in adoptive transfer systems (3 - 7). The nature of the memory B cell is controversial, however. The classic view postulates that these are cells which after primary stimulation by antigen do not differentiate into plasma cells, but become recirculating long-lived B lymphocytes, persisting in the system and giving rise to secondary responses upon a new encounter with antigen (8). Alternatively, it has been proposed that memory cells are short-lived and are either themselves in cell cycle or are the immediate progeny of cycling cells. Memory, in this view, is maintained either as persisting B cell clones of cycling primed cells (9,10) or by continuous recruitment of antigen-specific naive B cells from the bone marrow (11). Antgen, which often persists for a long time after immunization (12,13), and helper T cells would provide the selective forces driving new B cells into the 'memory pool'. In an attempt to discriminate between these models of memory maintenance we studied the dependence of B cell memory on T cell help. We show that depletion and/or blockade of helper T cells by chronic treatment of mice with anti-CD4 antibodies, for a period of time as long as 6 weeks, does not result in the loss of established memory in the B cell compartment. In accordance with earlier data (14-18), anti-CD4 treatment prevented the induction of primary and secondary responses to T celldependent antigens and also inhibited the generation of a
memory B cell compartment. This result suggests that the persistence of memory is independent of a continuous, T cell-dependent recruitment of memory B cells and may rely on T cell-independent mechanisms.
Methods Mice CBA/J adult female mice were used. The animals were either bred in our own animal colony or purchased from IFFA CREDO, L'Arbresle, France. Antigens and immunization (4-Hydroxy-3-nitrophenyl)acetyl (NP)-conjugated chicken yglobulin (NP-CG) was prepared as described previously (19). Mice were immunized i.p. with 100 mg of alum-precipitated NP-CG. Bordetella pertussis was used as adjuvant, injecting 2 x 108 organisms/mouse together with alum-precipitated NP-CG. Anti-CD4 antibodies for 'in vivo' treatment CD4 specific monoclonal antibodies GK1.5 (Rat lgG2b/x) (20) and 172.4 (Rat IgM/x) (21) were used. Chronic treatment of mice with these antibodies was done by i.p. injection of 200 /*g purified GK1.5/week and 400/xg 172.4 twice a week.
Correspondence to: K Rajewsky, Institute for Genetics, University of Cologne, WeyertaJ 121, D-5000 Koln 41, FRG Transmitting editor. I. L. Weissman
Received 22 December 1990, accepted 7 March 1990
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Abstract
488
Memory B cells persist under anti-CD4 treatment
Enzyme-linked immunosorbent assay (ELISA) To measure the concentration of antigen-specific antibodies in the sera of immunized mice an ELISA was performed as described earlier (22,23). Plates were coated with NIP - BSA and subsequently developed with biotinylated lg(4a)10.9 (specific for lgG1 of the a allotype. This antibody was a kind gift of Dr L A. Herzenberg). The assay was standardized with purified 18-1-16 (lgG1/X), an NP-specific antibody of BALB/c origin (24). Cell purification and transfer
Staining and fluorescence analysis Monoclonal anti-Ly1 (53-7.3) (27) and CD8-specific (53-6.7) (27) antibodies were purchased as biotin- and FITC-conjugates, respectively, from Becton Dickinson, Mountain View, CA. The CD4-specific monoclonal antibody GK1.5 was purified from cell line GK1.5 (20) and coupled to FITC. Cells were stained as described previously (28), with minor modifications. Briefly, 1 x 108 cells were washed in PBS containing 1 % BSA and 0.03% sodium azide and incubated with FITC- and biotjn-labeled antibodies at a concentration of 0.1 mg/ml for 30 mm on ice. After washing, the cells were incubated with streptavidin - PE (0.02 mg/ml) for another 30 min. They were then washed and resuspended at a final concentration of 2 x 106 cells/ml in medium containing propidium iodide. Fluorescence analysis was performed with a FACS 440 (Becton Dickinson, Mountain View, CA). To exclude dead cells from the analysis, the lymphocyte population was gated according to forward scatter versus propidium iodide staining. The data were analyzed with a Microvax using Electric Desk (29). Results Anti-CD4 treatment depletes most CD4 cells in the spleen Anti-CD4 antibodies, when injected in mice, eliminate most CD4+ cells in the peripheral lymphoid organs (15,16). We attempted to eliminate as many CD4+ cells as possible by injecting two rat monoclonal antibodies specific for mouse CD4 in combination. Mice were chronically treated with a mixture of antibody GK1.5 (lgG2b) and antibody 172.4 (IgM). After 1, 2, or 4 weeks of treatment their spleen cells were analyzed In order to identify the T cell subpopulations present, we stained the cells of treated mice with anti-Ly1 (as a pan-T cell marker), anti-CD4, and anti-CD8 (Fig. 1). Treatment with anti-CD4 antibodies under these conditions
Treatment with anti-CD4 inhibits primary antibody responses to NP - CG and does not induce tolerance Several studies have reported that anti-CD4-treated mice do not make primary responses to T cell-dependent antigens (14-18) Furthermore, in some cases antigen-priming of anti-CD4-treated mice induces tolerance to the immunizing antigen (32,33). In order to determine if this was the case using NP - CG as antigen, we injected mice with anti-CD4 antibodies on days - 1 , 0 , and + 1. On day 0 the mice were immunized with NP-CG. Five weeks later, at a time when 5 - 1 0 % CD4+ cells were present in the spleens of treated mice (not shown), the animals were re-immunized with NP-CG either as alum-precipitate or in soluble form. The serum titers of hapten-specific lgG1 antibodies on day 13 after primary immunization and on days 7 and 14 after secondary immunization are shown in Fig. 2. The anti-CD4 treatment inhibits the primary response to NP-CG, a finding common to all T cell-dependent antigenic systems tested (14-18). Five weeks after treatment the helper cell compartment has functionally recovered and is able to help the response to the same antigen if the latter is presented in a form immunogenic to naive mice (as alum-precipitate). We conclude that under treatment with anti-CD4 antibodies the immune system is blindfolded and ignores the antigen, becoming neither primed nor tolerant. Tolerance can be induced by simultaneous administration of antigen and anti-CD4 antibodies in some but not all cases (32,33). Anti-CD4 treatment inhibits the induction of the primary response but does not affect the ongoing immune response At which stages of the primary antibody response are CD4 + cells required? Mice were immunized with NP-CG and treatment with antj-CD4 was started after immunization in the early phases of the primary response. The kinetics of the responses obtained are shown in Fig. 3.
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B cells were purified from the spleens of donor mice by treatment with a monoclonal anti-Thy1.2 antibody (J1J; 25) and complement. The resulting cell populations contained < 1 % Thy1-positive cells as determined by flow cytometry. T cells were enriched from the spleens of donor mice over nylon wool columns (26). The nylon wool non-adherent cells contained 7 5 - 8 5 % Thy1-positive cells as judged by flow cytometry. Mixtures of T and B cells from donor mice were injected i.p. into syngeneic naive recipients that had been irradiated with 500 rad 1 day before cell transfer. The animals were boosted with 0.1 /iQ N P - C G on the day after transfer. The results are shown as geometric means with standard deviations of the titers of antigen-specific lgG1 antibodies in the sera of mice in each group.
results in the almost complete elimination of CD4+ cells from the spleen. A small population of 0.6-0.9% of cells are still present that are positive for CD4. After 1 week of treatment the percentage of CD8- T cells exceeds the percentage of CD4 + cells, suggesting the emergence of a small (2-3%) number of CD4-CD8- T cells. After longer periods of treatment (4 weeks) the percentage of cells with this phenotype does not exceed that seen in the control (1.1-1.5%) Ly1 B cells which also have this phenotype are distinguished from T cells because they are characteristically duller for the Ly1 marker (30,31). The CD4-CD8- T cells present in the initial period of treatment could derive from cells belonging to the CD4 lineage that had modulated the CD4 molecule or they could be cells that fail to stain with FITC-coupled GK1.5 because the determinant is blocked by the injected anti-CD4 antibody. We think that the latter explanation is unlikely because, in agreement with the results of others (16), we failed to detect cells staining with antibodies against rat Ig in treated mice (not shown). We have always observed the same degree of depletion of CD4+ cells in both the spleen and lymph nodes of mice treated with anti-CD4 antibodies (not shown). In contrast, no significant depletion of CD4 + cells in the thymus of treated mice was observed (not shown). This contrast between depletion in peripheral lymphoid organs and little or no effect in the thymus has also been reported earlier (16).
Memory B cells persist under anti-CD4 treatment 489
' ha
I
• 1
2
4
Fig. 1. Anti-CD4 treatment depletes most CD4 + T cells in the spleen. Staining of spleen cells from pools of three mice Ordinate (red fluorescence). Ly1, abscissa (green fluorescence)' CD8 (upper row), CD4 (lower row) Duration of anti-CD4 treatment and percentages of cells within the drfferent fluorescence windows are indicated in the figure
a-CD4 Group Treatment
2 Immunization 1-day 13
A
4-
10Ong Alum ppt.
2-day 7 2-day 15
B
10|ig soluble
1 -day 13 2'-day 7 2'-day 15
1'-day 13
soluble
10Oug Alum ppt.
2' -day 7 2"-day 15
±L
1 -day 13 2-day 7 2-day 15
10 10' anti-NIP lgG1
10
Fig. 2. Anti-CD4 treatment inhibits the primary response and does not induce tolerance to NP-CG. Groups of mice were either injected with anti-CD4 (groups A: • , and B: 0 ) or not treated (groups C: B ; and D' 82). All groups were immunized with NP-CG in alum (primary immunization). Five weeks later the mice were re-immunized (secondary immunization) with either alum-precipitated NP - CG (groups A and D) or soluble NP - CG (groups B and C). The figure shows geometric means and standard deviations of the triers of NIP-binding lgG1 antibodies in the sera of the mice in each group on the indicated days after primary or secondary immunization.
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Ti
CD4, FITC anti-CD4 treatment — (weeks)
490
Memory B cells persist under anti-CD4 treatment 10 3 -
i
Table 1. Anti-CD4 treatment prevents the generation of memory B cells
]
Hapten-binding IgGi G^g/ml)8
(9
O)
-I
2
10 -
I 10-
_
H8
15
_
.
5
8
21
28
—
_
•
«
.
_
8
35 days
Fig. 3. Anti-CD4 treatment does not lead to the abortion of the late primary response. Mice were either left untreated (O) or immunized with 100 tig NP - CG and not treated with anti-CD4 ( • ) Other groups were, in addition to the immunization, injected with anti-CD4 antibodies. • , mice injected from day 0 of immunization onwards; • , mice injected from day 5 after immunization, A, treatment started on day 11; T, treatment started on day 21. The serum titers of NIP-binding lgG1 antibodies of the mice in each of the groups on the indicated days after immunization are shown Groups of six mice were used.
No response is obtained if treatment is started on day 0 (that is, simultaneously with immunization), and only the early phase of the response (up to day 5) is sensitive to the removal and/or blockade of CD4+ cells. When treatment is started on day 11 or later the antibody response does not reach control values but is also not turned off. This result shows that it is only the induction of an immune response that requires a full helper T cell compartment. Established responses are less sensitive to anti-CD4 treatment and may not require helper T cells at all Memory induction requires an intact helper T cell compartment To determine whether memory B cells are induced under anti-CD4 treatment, mice were treated on days - 1 , 0 , and +1 (as in the experiment depicted in Fig. 2) and immunized on day 0 with NP-CG. Immunization was performed with alumprecipitated antigen as adjuvant and, in a separate experiment, with the addition of B.pertussis, because we found that priming using this adjuvant results in 5-to 10-fold higher titers in the secondary response (see below). Anti-CD4 treated, antigenprimed mice were then divided into two groups. Group 1 was chronically treated with anti-CD4 whereas in group 2 treatment was discontinued. Five weeks after immunization splenic B cells from each of these groups and also from control mice were mixed with T cells from carrier-primed mice and injected into irradiated recipients that were boosted with N P - C G 1 day after transfer. The titers of hapten-specific antibodies in the serum of the mice were determined and are shown in Table 1 In none of the groups of mice that were immunized under treatment could memory B cells be induced as measured in this assay. We conclude that, as was found to be the case with the
Experiment 1 b
Experiment 2
5 weeks (starting on day - 1 )
< 0 14
< 0 14
days - 1 , 0 , +1
< 0 14
< 0 14
_
9.9(1.4) < 0 14
51.3(1 5) < 0 14
"Shown are the geometric means with standard deviation coefficients of hapten-binding lgG1 serum antibody concentrations 0»g/ml) on day 12 after transfer of the 107 CG-primed T cells together with 107 B cells derived from mice of the respective groups (5 mice/group). b Pnming was done with alum-precipitated antigen (experiment 1) or alum-precipitated antigen plus B.pertussis (experiment 2) Cell transfer was performed 5 weeks after priming
primary antibody response, an intact CD4 compartment is necessary to induce memory B cells. An intact CD4+ T cell compartment is not necessary for the maintenance of memory in the B cell compartment We next asked whether established memory is lost following chronic treatment of mice with anti-CD4 antibodies. Mice were primed with NP-CG and B cell memory was measured 10 or 12 weeks later, by adoptive cell transfer. Before transfer, groups of these animals were treated with anti-CD4 for 1, 2,4, or 6 weeks. As in the previous experiments, two methods of priming were used. Mice primed with alum-precipitated antigen together with B.pertussis as adjuvant were treated for 4 or 6 weeks with anti-CD4 antibodies, and mice primed with alum-precipitated antigen only were treated for 1, 2, or 4 weeks. Splenic B cells from each of these groups, and also from control groups that had been either primed but not treated or not primed, were then transferred into irradiated hosts together with carrier-specific helper cells. The recipient mice were boosted with antigen, bled 12 days later and the anti-NP lgG1 titers in the sera were determined. The results are shown in Table 2. The response obtained from the groups of mice that received cells from anti-CD4-treated donors is indistinguishable from the control response. This is true regardless of the pnming conditions. The use of B.pertussis as adjuvant results in a much higher adoptive transfer response and therefore any loss in the ability to mount a secondary response should be more easily seen. The dependence of the response from the dose of B cells transferred shows that we were not working under saturating conditions. Therefore, loss of memory B cells should have been detectable, and we conclude from the experimental data that there is no measurable decay of memory in the B cell compartment under anti-CD4 treatment over a period of at least 6 weeks. Treatment with anti-CD4 does not eliminate all CD4+ cells (see Fig. 1). It was therefore important to estimate the level of help remaining in antigen-primed, anti-CD4-treated mice In a first experiment addressing this question we transferred the total
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1-
L __
Group 1, primed on day 0 Group 2, primed on day 0 Group 3, pnmed on day 0 Group 4, not pnmed
Anti-CD4 treatment
Memory B cells persist under anti-CD4 treatment
491
Table 2. Memory B cells persist under anti-CD4 treatment No. of B cells transferred Experiment 1 a 1 x 106 1 x 107
Experiment 2 2 x 106 1 x 107
Untreated 1 2 4 6
5 0 (1 3)" 6 0 (1 4) 4 0 (1 4) 7.2(1.7) nd
110 (1.2) nd n.d 93(2 7) 245 (1.8)
1.5(1.7) 1 4 (2.5) 0.9(1.9) 1 2 (1 9) n.d
17 (3.0) nd nd 48 (1 5) 22 (3 9)
10-
1-
0
I
7
10/tg NP-CG
B 100-
• •
o at
!
D
•
10-
1-
B
B
• •D
a
a
D
a
• •
a
0.10
1 2
4
6
weeks of anti-CD4 treatment Fig. 4. Chronic anti-CD4 treatment impairs the ability to mount an adoptive transfer response. Groups of mice received 2 x 107 spleen cells from nonpnmed mice (A) or from mice primed with NP-CG and later treated for the indicated lengths of time with anti-CD4 ( • , • ) Closed symbols' pnming with alum-precipitated antigen plus B.pertussis. Open symbols, pnming with alum-precipitated antigen only. The figure shows the trters of antigen-specific antibodies in the serum of individual mice on day 12 after boosting
spleen cells from the mice that had been primed and treated as described in the previous experiment. Since under the conditions of that experiment B cells from anti-CD4-treated and control animals produce indistinguishable adoptive responses when sufficient T cell help is provided (Table 2), any impairment in the response obtained from total spleen cells would be due to lack of T cell help The result of this experiment is shown in Fig. 4, where the titers from individual mice are plotted. Clearly, helper
14
30 days
Fig. 5. Anti-CD4 treatment inhibits the in situ secondary response Mice were immunized on day 0 with 10 /ig NP - CG without adjuvant. Serum trters of napten-specrtlc lgG1 antibodies in the serum before immunization and on days 7, 14, and 30 are shown • , unpnmed mice; O, mice that had been primed with NP-CG 10 weeks previously, • , mice that had been primed with NP-CG 10 weeks before and then were injected with anti-CD4 antibodies for 1 week before immunization Six mice in each group activity can be detected in the adoptive secondary response after anti-CD4 treatment of the donors. Some loss of help seems to occur with time, as longer periods of treatment result in somewhat lower adoptive responses spread over a wide range, with some mice failing to respond at all. Taken together, the results of this experiment indicate that anti-CD4 treatment does not completely eliminate CD4-positive T cells from the animal. Possibly the C D 4 - C D 8 - T cells present in the spleen in the early period of treatment (Fig. 1) belong to the CD4 lineage, and after transfer into nontreated recipients are able to expand and/or re-express the CD4 molecule and thus help the memory B cells present to make a response. With longer periods of treatment more and more of these cells are eliminated, resulting, after adoptive transfer, in a helper activity that is less and less efficient Because of this experimental result it became mandatory to assess helper activity in the anti-CD4-treated animals more directly. This was done by analyzing the capacity of the manipulated animals to mount a secondary response in situ, in the presence of anti-CD4 antibodies. In accord with earlier data (14,15), a secondary response could not be elicited in this situation (Fig. 5) Inhibition amounts to a factor of 10 at least; background levels of serum antibodies do not permit us to determine whether the reduction is even larger. The absence of a secondary response in these mice could be due to suppression of the antigen-specific response. This seems unlikely as after recovery from anti-CD4 treatment the mice are able to mount an anti-NP immune response (see Fig. 2). Our interpretation of these results is that in the anti-CD4-treated mice the circulating anti-CD4 antibodies block the accessory molecule (or lead to its
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n d , not determined "Priming was done with alum-precipitated antigen (experiment 1) or alum-precipitated antigen plus B.pertussis (experiment 2). Cefl transfer was performed 10 (experiment 1) or 12 weeks (experiment 2) after pnming bGeometric means with standard deviation coefficients of haptenbmding lgG1 serum antibody concentration (^g/ml) on day 12 after transfer of 107 CG-pnmed T cells together with the indicated number of B cells from NP - CG-pnmed mice which were treated with anti-CD4 for the last 1, 2, 4, or 6 weeks before transfer as indicated (mean of 3 - 5 mice/group). Serum trters of mice that received 107 CG-pnmed T cells plus either 107 B cells from nave donors or 107 B cells from primed donors but were not boosted with antigen after transfer were < 0 14 /ig/ml.
s anti-NIF
Anti-CD4 treatment (weeks)
492
Memory B cells persist under anti-CD4 treatment
3:
10-
c
1-
CD
0.1 5
8 11 14
21
28 days
modulation) in the cells that are not eliminated, thereby rendering them unable to deliver helper signals to B cells. Very few if any functional CD4+ helper cells remain under treatment. To generate a memory T cell compartment CD4+ cells are needed for the first 3 weeks after immunization If our conclusion that treatment of mice with anti-CD4 antibodies inhibits the generation of memory B cells but does not affect established memory is correct, then this system of anti-CD4 treatment should allow an estimate of the time that is necessary to generate a full memory B cell compartment after immunization. Groups of antigen-primed mice were treated with anti-CD4, starting at different times after immunization. B cells from each of these groups were taken on day 35 after priming and transferred together with carrier-specific helper cells into irradiated recipients that were then boosted with antigen. The antibody trters in the sera of the recipient mice on day 12 after boosting are shown in Fig. 6. The result shows that the groups in which treatment was started less than 21 days after immunization did not generate a full memory compartment; only after day 21 is the adoptive response indistinguishable from the control. This result is compatible with previous estimates of 2 - 4 weeks as the time necessary to generate a full memory compartment (1,34,35). It supports our conclusion that CD4 + cells are necessary to induce memory, but that whatever memory is already present is not lost upon treatment with anti-CD4 antibodies. Discussion In this work we investigated the effect of blocking helper T cell function at different stages of the immune response to a T cell-dependent antigen. Treatment of mice with anti-CD4 antibodies eliminates most CD4+ cells from peripheral lymphoid organs (Fig 1). The inhibition of the primary and secondary antibody responses (Figs
An alternative interpretation is that the circulating anti-CD4 antibodies do not block the few CD4+ cells that remain. These may represent a population of primed cells whose responses have higher affinity to antigen. This would make the interaction between primed CD4 + cells and B cells less sensitive to blocking of the accessory molecule. This interpretation is contradicted by the finding that the secondary response (where primed T cells are already present) is also inhibited by treatment with anti-CD4 (Fig. 5). If primed T cells were able to help B cells in treated mice one would not expect the secondary response to be inhibited by anti-CD4 treatment. One might thus resort to the possibility that an interaction of primed helper T with memory B cells in the phase of memory maintenance may take place in a 'protected' environment where the concentration of CD4-specific antibodies is not high enough to block the interaction of the CD4 molecule with its target or to eliminate the helper cells altogether The thymus may represent such an environment (16), but is hardly the site of B cell memory maintenance. Another microenvironment which could be considered in this context would be the germinal centers which, like the thymus, represent dense lymphocyte accumulations. However, germinal centers form in the phase of B cell memory induction (which is inhibitable by anti-CD4 antibodies) and disappear thereafter (38). Thus, the simplest interpretation of the present data is that antigen priming results in the generation of memory B cells that are long-lived and persist in the organism, able to mount a secondary antibody response if helper T cells are available. The late primary antibody response (and the secondary response) would be produced by long-lived plasma cells generated after immunization, in parallel with the generation of memory B cells Long-lived, IgG-secreting plasma cells exist in the bone marrow (39,40). It has also been found that plasma cells which migrate to the bone marrow appear late in the primary response and in secondary responses (41,42). If this interpretation is correct, one would predict that memory B cells are also antigen-independent, requiring antigen only for the initiation of the response. Evidence to the contrary obtained in mice (1,4), and recently reproduced in rats (43), could be the
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Fig. 6. Anti-CD4 treatment interrupts the build up of B cell memory. Groups of mice received 107 cells from CG-primed mice together with 107 B cells from. A , unpnmed mice, • , mice that were primed with antigen and then chronically treated with anti-CD4 starting on the indicated days after immunization, • , antigen-pnmed nontreated mice All mice were boosted 1 day after cell transfer and their serum responses measured on day 12 after boost. NIP+71 + responses are shown (5 mice/group)
2 and 5) and of the induction of B cell memory (Table 1) shows that CD4+ cells are required for these processes However, this is true only for the early phase after immunization. When treatment with anti-CD4 is delayed until 3 weeks or more after immunization little or no effect is observed: the ongoing antibody response is unaffected (Fig. 3) and there is no detectable loss of B cell memory (Table 2). We interpret these results as suggesting that the maintenance of both an ongoing antibody response and of memory in the B cell compartment are helper T cell independent. This interpretation rests on the assumption that anti-CD4 treatment effectively blocks all helper T cell-dependent mechanisms, preventing the few CD4+ cells remaining under treatment from delivering help to B cells. Treatment with anti-CD4 antibodies may inhibit helper T cell-dependent functions not only by eliminating most CD4+ cells but also by leading to the modulation of the accessory molecule and directly blocking the cells that remain. All of this would result in the complete inhibition of CD4+ T cell - B cell interactions. Supporting this view is the finding that treatment of mice with Fab fragments of CD4-specific antibodies fully inhibits the response to T cell-dependent antigens with minimal depletion of CD4+ cells (36,37).
Memory B cells persist under anti-CD4 treatment
Whatever the mechanism is that leads to the maintenance of memory in the B cell compartment, the fact that the amount of help (if any) needed to maintain memory B cells is less than that necessary to stimulate naive B cells and drive them into the memory pathway argues against the idea that a continuous recruitment of naive B cells (11) is the major mechanism of perpetuation of immunological memory to T celldependent antigens.
Acknowledgements We are grateful to Irmgard Forster for discussion, advice, and help with the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft through SFBs 74 and 243, and by the Fazrt Foundation. Abbreviations CG ELISA NP
chicken -^-globulin enzyme-linked immunosorbent assay (4-hydroxy-3-nrtropheny()acetyl
References 1 Celada, F. 1971 The cellular basis of immunologic memory Progr Allergy 15 223. 2 Kocks, C and Ra/ewsky, K. 1989. Stable expression and somatic hypermutation of antibody V regions in B cell differentiation pathways Annu. Rev. Immunol. 7:537 3 Celada, F. 1966. Quantitative studies of the adoptive immunological memory in mice. I: An age dependent barrier to syngenetc transplantation. J. Exp. Med. 124.1. 4 Celada, F. 1967 Quantitative studies of the adoptive immunotogical memory in mice. II: Linear transmission of cellular memory. J. Exp. Med. 125199. 5 Mitchison, N. A. 1971 The carrier effect in the secondary response to hapten - protein conjugates. I: Measurement of the effect with transfected cells and objections to the local environment hypothesis Eur. J. Immunol. 1:10. 6 Mitchison, N. A. 1971. The carrier effect in the secondary response to hapten - protein conjugates. II: Cellular cooperation. Eur J
Immunol. 1:18. 7 Stekevrtz, M , Kocks, C . Fiajewsky, K., and Dildrop, R. 1987. Analysts of somatic mutation and class switching in naive and memory B cells generating adoptive primary and secondary responses. Cell 48757 8 Strober, S. 1975. Immune function, cell surface characteristics and maturation of B cell subpopulations. Transplant. Rev 24:84. 9 Gray, D., MacLennan, I. C. M., and Lane, P. J. L. 1986. Virgin B cell recruitment and the lifespan of memory clones dunng antibody responses to 2,4-dinitrophenylhemocyanin Eur. J. Immunol. 16:641. 10 Zhang, J , Liu, Y.-J , MacLennan, I. C. M., Gray, D., and Lane, P. J. L. 1988 B cell memory to thymus-independent antigens type 1 and 2the role of lipopotysaccharide in B memory induction Eur. J. Immunol. 18:1417. 11 Code, J.-H., Truffa-Bacchi, P., and Freitas, A. A. 1988. Secondary antibody responses to thymus independent antigens. Decline and life-span of memory. Eur J. Immunol 18-1307. 12 Tew J. G., Mandel, T. E , and Burgess, A. W. 1979 Retention of intact HSA for prolonged periods in the popliteal lymph nodes of specifically immunized mice. Cell. Immunol 45 207. 13 Tew, J. G. and Mandel, T. E 1979. Prolonged antigen half-life in the lymphoid follicles of specifically immunized mice. Immunology 37.69 14 Coulie, P. G., Coutelier, J.-P, Uyttenhove, C , Lambotte, P., and Van Snick, J. 1985. In vivo suppression of T dependent antibody responses by treatment with a monoclonal anti-L3T4 antibody. Eur. J. Immunol 15.638. 15 Goronzy, J., Weynand, C M . , and Fathman, C G. 1986. Long-term humoral unresponsiveness in vivo induced by treatment with monoclonal antibody against L3T4. J. Exp. Med. 164911. 16 Wofsy, D., Mayes, D. C , Woodcock, J , and Seaman, W. E. 1985. Inhibition of humoral immunity in vivo by monoclonal antibody to L3T4: studies with soluble antigens in intact mice. J. Immunol 135:1698. 17 Bobbdd, S. P., Jayasuriya, A., Nash, A , Prospero, T D, and Waldman, H. 1984 Therapy with monoclonal antibodies by elimination of T cell subsets in vivo. Nature 312.548. 18 Qin, S., Cobbold, S , Tighe, H , Benjamin, R., and Waldman, H. 1987. CD4 monoclonal antibody pairs for immunosuppressKjn and tolerance induction. Eur. J Immunol 17-1159 19 Imanishi, T. and Makela, O. 1973. Strain differences in the fine specificity of mouse anti-hapten antibodies. Eur. J. Immunol 3323 20 Dialynas, D. P , Quan, Z. S., Wall, K. A., Pierres, A., Quintans, J., Loken, M. R., Pierres, M., and Fitch, F W 1983. Characterization of the murine T cell surface molecule designated L3T4, identified by monoclonal antibody GK1.5: similarity of L3T4 to the human Leu3/T4 molecule. J. Immunol. 131.2445. 21 Ceredig, R., Lowenthal, J. W., Nabholz, M , and MacDonald, H. R. 1985. Expression of interleukin 2 receptors as a differentiation marker on intrathymic stem cells. Nature 31498. 22 Kendall, C , lonescu-Matiu, I., and Dreesman, G. R 1984. Utilization of the biotin/avidin system to amplify the sensitivity of the Enzyme Linked Immunosorbent Assay J. Immunol. Method 56:329. 23 Vieira, P. and Rajewsky, K. 1988. The half-lives of serum immunoglobulin in adult mice. Eur. J. Immunol. 18313. 24 White-Scharff, M and Imanishi-Kari, T. 1981. Characterization of the NPa idiotype through analysis of monoclonal BALB/c anti-(4-hydroxy3-nitrophenyl)acetyl (NP) antibodies. Eur. J. Immunol. 11:897. 25 Bruce, J., Symington, F. W., McKearn, T J., and Sprent, J. 1981 A monoclonal antibody discrimination between subsets of T and B cells. J Immunol. 127:2496 26 Julius, M. H., Simpson, E , and Herzenberg, L. A. 1973. A rapid method for the isolation of functional thymus-derived murine lymphocytes. Eur. J. Immunol. 3:645. 27 Ledbetter, J. A. and Herzenberg, L. A. 1979. Xenogenac monoclonal antibodies to mouse lymphoid differentiation antigens. Immunol Rev 47:63. 28 Weichel, W., Liesegang, B., Gehrke, K., Gottiinger, C , Hottkamp, B., Radbruch, A., Stackhouse, T. K., and Rajewsky, K. 1985. Inexpensive upgrading of a FACS I: isolation of rare somatic variants by doublefluorescence sorting. Cytometry 6:116. 29 Moore, W. A. and Kautz, R. A. 1986. Data analysis in flow cytometry. In War, D M., ed., Handbook of Experimental Immunology, Vol. I, p 30.1. Blackwell Scientific, Oxford. 30 Hayakawa, K., Hardy, R., Parks, D. R., and Herzenberg, L. A. 1983. The 'Ly 1 B' cell subpopulation in normal, jmmunodefective and
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result of the cell transfer system used, and does not directly address the question of antigen dependency of memory B cells because the loss of memory responses observed after transfer of cells into irradiated, non-primed animals could be due to lack of memory T cells or to cellular competition in the repopulation of the host's immune system. Since at this point one cannot totally exclude that in antigenprimed, anti-CD4-treated mice some helper T cells are functional and able to deliver help to B cells, we believe that the minimal conclusion from the experiments reported here is that different amounts of T cell help are needed at different stages of the response to T cell-dependent antigens. Early stages of immune responses to antigen are strictly helper T cell-dependent and are fully inhibited by anti-CD4 treatment; later stages are less or not at all dependent on help from CD4+ T cells. The former include the induction of the primary and secondary responses (14-18)— the functions classically used to define thymus(helper)-dependent antigens and responses (44-47). Our work demonstrates that the generation of a memory B cell compartment belongs also to this category. Events that take place after the induction of the immune response, like the persistence of memory in the B cell compartment and the maintenance of an ongoing primary antibody response, require less or no help from CD4+ cells.
493
494
Memory B cells persist under anti-CD4 treatment 39 Hibi, T. and Dosch, H. M 1986 Limiting dilution analysis of the B cell compartment in human bone marrow. Eur J Immunol 16 139 40 Ho, F., Lortan, J. E., MacLennan, I. C M., and Khan, M 1986 Distinct short-lived and long-lived antibody-producing cell populations. Eur J. Immunol. 16:1297. 41 Benner, R., Hijmans, W., and Haaijman, J J. 1981. The bone marrow, the major source of serum immunoglobtilins but still a neglected site of antibody formation Clin. Exp Immunol. 46 1. 42 Benner, R., van Oudena/en, A., and Koch, G 1981. Induction of antibody formation in mouse bone marrow In Lefkovitz, I and Pernis, B , eds, Immunological Methods, Vol. II, p. 247. Academic Press, New York. 43 Gray, D. and Skavall, H 1988 B cell memory is short-lived in the absence of antigen Nature 336:70 44 Miller, J. F. A. P. and Mitchell, G F. 1969. Thymus and antigen-reactive cells Transplant Rev. 13. 45 Taylor, R. B 1969. Cellular cooperation in the antibody response of mice to two serum albumins, specific function of thymus cells. Transplant. Rev. 1114 46 Raff, M C 1970. Role of thymus-denved lymphocytes in the secondary humoral immune response in mice. Nature 2261257. 47 Kindred, B. 1971. Immunological unresponsiveness of genetically thymusless (nude) mce. Eur. J. Immunol 1 59.
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autoimmune mice. J. Exp Med. 157202. 31 Forster, I and Rajewsky, K. 1987. Expansion and functional activity of Ly1 + B cells upon transfer of peritoneal cells into allotypecongenic newborn mice. Eur J. Immunol 17:521 32 Benjamin, R. J. and Waldman, H. 1986. Induction of tolerance by monoclonal antibody therapy. Nature 320 449 33 Gutstein, N. L, Seaman, W E , Scott, J. H., and Wofsy, D 1986 Induction of immune tolerance by administration of monoclonal antibody to L3T4. J Immunol. 137.1127. 34 Herzenberg, L A , Black, S J , Tokuhias, T., and Herzenberg, L. A. 1980. Memory B cells at successive stages of differentiation. Affinity maturation and the role of IgD receptors J Exp. Med. 151:1071 35 Cerottini, J.-C and Trnka, Z. 1970 The role of persisting antigen in the development of immunological memory. Int. Arch. Allergy 38:37 36 Gutstein, N L. and Wofsy, D 1986 Administration of F(abO2 fragments of monoclonal antibody to L3T4 inhibits humoral immunity in mice without depleting L3T4+ cells. J. Immunol. 137 3414. 37 Carteron, N. L, Wofsy, D., and Seaman, W. E 1988. Induction of immune tolerance during administration of monoclonal antibody to L3T4 does not depend on depletion of L3T4+ cells J Immunol. 140713 38 Nieuwenhuis, P. and Opstelten, D. 1984 Functional anatomy of germinal centres Am. J. Anat 170421
© 1990 Oxford University Press 0953-8178/90 $3.00
Persistence of memory B cells in mice deprived of T cell help Paulo Vieira1 and Klaus Rajewsky Institute for Genetics, University of Cologne, FRG 'Present address. DNAX, Palo Alto, CA 94304-1104, USA Key words: memory B cells, B cell memory, T cell help
The influence of T cell help on the Induction, maintenance, and recall of B cell memory to a T cell-dependent antigen was studied In mice. The antigen was a hapten coupled to a protein carrier, and helper T cells were eliminated before, during, or after immunization by treatment of the animals with antl-CD4 antibodies. B cell memory was quantified in an adoptive cell transfer system, transferring B cells from the primed animals together with carrier-primed syngeneic T cells. Antl-CD4 treatment completely inhibited the induction of primary and secondary responses and of B cell memory. In contrast, it did not affect established B cell memory over a period of at least 6 weeks. Consequently, this suggests that within this time range B cell memory to a T celldependent antigen does not rely on the continuous recruitment of naive B cells Into the memory compartment, and memory B cells need little or no (antigen-mediated) T cell help In order to persist. Introduction Memory B cells are usually defined as cells which make secondary responses to thymus-dependent antigens (1,2). They easily giveriseto antibody responses in adoptive transfer systems (3 - 7). The nature of the memory B cell is controversial, however. The classic view postulates that these are cells which after primary stimulation by antigen do not differentiate into plasma cells, but become recirculating long-lived B lymphocytes, persisting in the system and giving rise to secondary responses upon a new encounter with antigen (8). Alternatively, it has been proposed that memory cells are short-lived and are either themselves in cell cycle or are the immediate progeny of cycling cells. Memory, in this view, is maintained either as persisting B cell clones of cycling primed cells (9,10) or by continuous recruitment of antigen-specific naive B cells from the bone marrow (11). Antgen, which often persists for a long time after immunization (12,13), and helper T cells would provide the selective forces driving new B cells into the 'memory pool'. In an attempt to discriminate between these models of memory maintenance we studied the dependence of B cell memory on T cell help. We show that depletion and/or blockade of helper T cells by chronic treatment of mice with anti-CD4 antibodies, for a period of time as long as 6 weeks, does not result in the loss of established memory in the B cell compartment. In accordance with earlier data (14-18), anti-CD4 treatment prevented the induction of primary and secondary responses to T celldependent antigens and also inhibited the generation of a
memory B cell compartment. This result suggests that the persistence of memory is independent of a continuous, T cell-dependent recruitment of memory B cells and may rely on T cell-independent mechanisms.
Methods Mice CBA/J adult female mice were used. The animals were either bred in our own animal colony or purchased from IFFA CREDO, L'Arbresle, France. Antigens and immunization (4-Hydroxy-3-nitrophenyl)acetyl (NP)-conjugated chicken yglobulin (NP-CG) was prepared as described previously (19). Mice were immunized i.p. with 100 mg of alum-precipitated NP-CG. Bordetella pertussis was used as adjuvant, injecting 2 x 108 organisms/mouse together with alum-precipitated NP-CG. Anti-CD4 antibodies for 'in vivo' treatment CD4 specific monoclonal antibodies GK1.5 (Rat lgG2b/x) (20) and 172.4 (Rat IgM/x) (21) were used. Chronic treatment of mice with these antibodies was done by i.p. injection of 200 /*g purified GK1.5/week and 400/xg 172.4 twice a week.
Correspondence to: K Rajewsky, Institute for Genetics, University of Cologne, WeyertaJ 121, D-5000 Koln 41, FRG Transmitting editor. I. L. Weissman
Received 22 December 1990, accepted 7 March 1990
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Abstract
488
Memory B cells persist under anti-CD4 treatment
Enzyme-linked immunosorbent assay (ELISA) To measure the concentration of antigen-specific antibodies in the sera of immunized mice an ELISA was performed as described earlier (22,23). Plates were coated with NIP - BSA and subsequently developed with biotinylated lg(4a)10.9 (specific for lgG1 of the a allotype. This antibody was a kind gift of Dr L A. Herzenberg). The assay was standardized with purified 18-1-16 (lgG1/X), an NP-specific antibody of BALB/c origin (24). Cell purification and transfer
Staining and fluorescence analysis Monoclonal anti-Ly1 (53-7.3) (27) and CD8-specific (53-6.7) (27) antibodies were purchased as biotin- and FITC-conjugates, respectively, from Becton Dickinson, Mountain View, CA. The CD4-specific monoclonal antibody GK1.5 was purified from cell line GK1.5 (20) and coupled to FITC. Cells were stained as described previously (28), with minor modifications. Briefly, 1 x 108 cells were washed in PBS containing 1 % BSA and 0.03% sodium azide and incubated with FITC- and biotjn-labeled antibodies at a concentration of 0.1 mg/ml for 30 mm on ice. After washing, the cells were incubated with streptavidin - PE (0.02 mg/ml) for another 30 min. They were then washed and resuspended at a final concentration of 2 x 106 cells/ml in medium containing propidium iodide. Fluorescence analysis was performed with a FACS 440 (Becton Dickinson, Mountain View, CA). To exclude dead cells from the analysis, the lymphocyte population was gated according to forward scatter versus propidium iodide staining. The data were analyzed with a Microvax using Electric Desk (29). Results Anti-CD4 treatment depletes most CD4 cells in the spleen Anti-CD4 antibodies, when injected in mice, eliminate most CD4+ cells in the peripheral lymphoid organs (15,16). We attempted to eliminate as many CD4+ cells as possible by injecting two rat monoclonal antibodies specific for mouse CD4 in combination. Mice were chronically treated with a mixture of antibody GK1.5 (lgG2b) and antibody 172.4 (IgM). After 1, 2, or 4 weeks of treatment their spleen cells were analyzed In order to identify the T cell subpopulations present, we stained the cells of treated mice with anti-Ly1 (as a pan-T cell marker), anti-CD4, and anti-CD8 (Fig. 1). Treatment with anti-CD4 antibodies under these conditions
Treatment with anti-CD4 inhibits primary antibody responses to NP - CG and does not induce tolerance Several studies have reported that anti-CD4-treated mice do not make primary responses to T cell-dependent antigens (14-18) Furthermore, in some cases antigen-priming of anti-CD4-treated mice induces tolerance to the immunizing antigen (32,33). In order to determine if this was the case using NP - CG as antigen, we injected mice with anti-CD4 antibodies on days - 1 , 0 , and + 1. On day 0 the mice were immunized with NP-CG. Five weeks later, at a time when 5 - 1 0 % CD4+ cells were present in the spleens of treated mice (not shown), the animals were re-immunized with NP-CG either as alum-precipitate or in soluble form. The serum titers of hapten-specific lgG1 antibodies on day 13 after primary immunization and on days 7 and 14 after secondary immunization are shown in Fig. 2. The anti-CD4 treatment inhibits the primary response to NP-CG, a finding common to all T cell-dependent antigenic systems tested (14-18). Five weeks after treatment the helper cell compartment has functionally recovered and is able to help the response to the same antigen if the latter is presented in a form immunogenic to naive mice (as alum-precipitate). We conclude that under treatment with anti-CD4 antibodies the immune system is blindfolded and ignores the antigen, becoming neither primed nor tolerant. Tolerance can be induced by simultaneous administration of antigen and anti-CD4 antibodies in some but not all cases (32,33). Anti-CD4 treatment inhibits the induction of the primary response but does not affect the ongoing immune response At which stages of the primary antibody response are CD4 + cells required? Mice were immunized with NP-CG and treatment with antj-CD4 was started after immunization in the early phases of the primary response. The kinetics of the responses obtained are shown in Fig. 3.
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B cells were purified from the spleens of donor mice by treatment with a monoclonal anti-Thy1.2 antibody (J1J; 25) and complement. The resulting cell populations contained < 1 % Thy1-positive cells as determined by flow cytometry. T cells were enriched from the spleens of donor mice over nylon wool columns (26). The nylon wool non-adherent cells contained 7 5 - 8 5 % Thy1-positive cells as judged by flow cytometry. Mixtures of T and B cells from donor mice were injected i.p. into syngeneic naive recipients that had been irradiated with 500 rad 1 day before cell transfer. The animals were boosted with 0.1 /iQ N P - C G on the day after transfer. The results are shown as geometric means with standard deviations of the titers of antigen-specific lgG1 antibodies in the sera of mice in each group.
results in the almost complete elimination of CD4+ cells from the spleen. A small population of 0.6-0.9% of cells are still present that are positive for CD4. After 1 week of treatment the percentage of CD8- T cells exceeds the percentage of CD4 + cells, suggesting the emergence of a small (2-3%) number of CD4-CD8- T cells. After longer periods of treatment (4 weeks) the percentage of cells with this phenotype does not exceed that seen in the control (1.1-1.5%) Ly1 B cells which also have this phenotype are distinguished from T cells because they are characteristically duller for the Ly1 marker (30,31). The CD4-CD8- T cells present in the initial period of treatment could derive from cells belonging to the CD4 lineage that had modulated the CD4 molecule or they could be cells that fail to stain with FITC-coupled GK1.5 because the determinant is blocked by the injected anti-CD4 antibody. We think that the latter explanation is unlikely because, in agreement with the results of others (16), we failed to detect cells staining with antibodies against rat Ig in treated mice (not shown). We have always observed the same degree of depletion of CD4+ cells in both the spleen and lymph nodes of mice treated with anti-CD4 antibodies (not shown). In contrast, no significant depletion of CD4 + cells in the thymus of treated mice was observed (not shown). This contrast between depletion in peripheral lymphoid organs and little or no effect in the thymus has also been reported earlier (16).
Memory B cells persist under anti-CD4 treatment 489
' ha
I
• 1
2
4
Fig. 1. Anti-CD4 treatment depletes most CD4 + T cells in the spleen. Staining of spleen cells from pools of three mice Ordinate (red fluorescence). Ly1, abscissa (green fluorescence)' CD8 (upper row), CD4 (lower row) Duration of anti-CD4 treatment and percentages of cells within the drfferent fluorescence windows are indicated in the figure
a-CD4 Group Treatment
2 Immunization 1-day 13
A
4-
10Ong Alum ppt.
2-day 7 2-day 15
B
10|ig soluble
1 -day 13 2'-day 7 2'-day 15
1'-day 13
soluble
10Oug Alum ppt.
2' -day 7 2"-day 15
±L
1 -day 13 2-day 7 2-day 15
10 10' anti-NIP lgG1
10
Fig. 2. Anti-CD4 treatment inhibits the primary response and does not induce tolerance to NP-CG. Groups of mice were either injected with anti-CD4 (groups A: • , and B: 0 ) or not treated (groups C: B ; and D' 82). All groups were immunized with NP-CG in alum (primary immunization). Five weeks later the mice were re-immunized (secondary immunization) with either alum-precipitated NP - CG (groups A and D) or soluble NP - CG (groups B and C). The figure shows geometric means and standard deviations of the triers of NIP-binding lgG1 antibodies in the sera of the mice in each group on the indicated days after primary or secondary immunization.
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Ti
CD4, FITC anti-CD4 treatment — (weeks)
490
Memory B cells persist under anti-CD4 treatment 10 3 -
i
Table 1. Anti-CD4 treatment prevents the generation of memory B cells
]
Hapten-binding IgGi G^g/ml)8
(9
O)
-I
2
10 -
I 10-
_
H8
15
_
.
5
8
21
28
—
_
•
«
.
_
8
35 days
Fig. 3. Anti-CD4 treatment does not lead to the abortion of the late primary response. Mice were either left untreated (O) or immunized with 100 tig NP - CG and not treated with anti-CD4 ( • ) Other groups were, in addition to the immunization, injected with anti-CD4 antibodies. • , mice injected from day 0 of immunization onwards; • , mice injected from day 5 after immunization, A, treatment started on day 11; T, treatment started on day 21. The serum titers of NIP-binding lgG1 antibodies of the mice in each of the groups on the indicated days after immunization are shown Groups of six mice were used.
No response is obtained if treatment is started on day 0 (that is, simultaneously with immunization), and only the early phase of the response (up to day 5) is sensitive to the removal and/or blockade of CD4+ cells. When treatment is started on day 11 or later the antibody response does not reach control values but is also not turned off. This result shows that it is only the induction of an immune response that requires a full helper T cell compartment. Established responses are less sensitive to anti-CD4 treatment and may not require helper T cells at all Memory induction requires an intact helper T cell compartment To determine whether memory B cells are induced under anti-CD4 treatment, mice were treated on days - 1 , 0 , and +1 (as in the experiment depicted in Fig. 2) and immunized on day 0 with NP-CG. Immunization was performed with alumprecipitated antigen as adjuvant and, in a separate experiment, with the addition of B.pertussis, because we found that priming using this adjuvant results in 5-to 10-fold higher titers in the secondary response (see below). Anti-CD4 treated, antigenprimed mice were then divided into two groups. Group 1 was chronically treated with anti-CD4 whereas in group 2 treatment was discontinued. Five weeks after immunization splenic B cells from each of these groups and also from control mice were mixed with T cells from carrier-primed mice and injected into irradiated recipients that were boosted with N P - C G 1 day after transfer. The titers of hapten-specific antibodies in the serum of the mice were determined and are shown in Table 1 In none of the groups of mice that were immunized under treatment could memory B cells be induced as measured in this assay. We conclude that, as was found to be the case with the
Experiment 1 b
Experiment 2
5 weeks (starting on day - 1 )
< 0 14
< 0 14
days - 1 , 0 , +1
< 0 14
< 0 14
_
9.9(1.4) < 0 14
51.3(1 5) < 0 14
"Shown are the geometric means with standard deviation coefficients of hapten-binding lgG1 serum antibody concentrations 0»g/ml) on day 12 after transfer of the 107 CG-primed T cells together with 107 B cells derived from mice of the respective groups (5 mice/group). b Pnming was done with alum-precipitated antigen (experiment 1) or alum-precipitated antigen plus B.pertussis (experiment 2) Cell transfer was performed 5 weeks after priming
primary antibody response, an intact CD4 compartment is necessary to induce memory B cells. An intact CD4+ T cell compartment is not necessary for the maintenance of memory in the B cell compartment We next asked whether established memory is lost following chronic treatment of mice with anti-CD4 antibodies. Mice were primed with NP-CG and B cell memory was measured 10 or 12 weeks later, by adoptive cell transfer. Before transfer, groups of these animals were treated with anti-CD4 for 1, 2,4, or 6 weeks. As in the previous experiments, two methods of priming were used. Mice primed with alum-precipitated antigen together with B.pertussis as adjuvant were treated for 4 or 6 weeks with anti-CD4 antibodies, and mice primed with alum-precipitated antigen only were treated for 1, 2, or 4 weeks. Splenic B cells from each of these groups, and also from control groups that had been either primed but not treated or not primed, were then transferred into irradiated hosts together with carrier-specific helper cells. The recipient mice were boosted with antigen, bled 12 days later and the anti-NP lgG1 titers in the sera were determined. The results are shown in Table 2. The response obtained from the groups of mice that received cells from anti-CD4-treated donors is indistinguishable from the control response. This is true regardless of the pnming conditions. The use of B.pertussis as adjuvant results in a much higher adoptive transfer response and therefore any loss in the ability to mount a secondary response should be more easily seen. The dependence of the response from the dose of B cells transferred shows that we were not working under saturating conditions. Therefore, loss of memory B cells should have been detectable, and we conclude from the experimental data that there is no measurable decay of memory in the B cell compartment under anti-CD4 treatment over a period of at least 6 weeks. Treatment with anti-CD4 does not eliminate all CD4+ cells (see Fig. 1). It was therefore important to estimate the level of help remaining in antigen-primed, anti-CD4-treated mice In a first experiment addressing this question we transferred the total
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1-
L __
Group 1, primed on day 0 Group 2, primed on day 0 Group 3, pnmed on day 0 Group 4, not pnmed
Anti-CD4 treatment
Memory B cells persist under anti-CD4 treatment
491
Table 2. Memory B cells persist under anti-CD4 treatment No. of B cells transferred Experiment 1 a 1 x 106 1 x 107
Experiment 2 2 x 106 1 x 107
Untreated 1 2 4 6
5 0 (1 3)" 6 0 (1 4) 4 0 (1 4) 7.2(1.7) nd
110 (1.2) nd n.d 93(2 7) 245 (1.8)
1.5(1.7) 1 4 (2.5) 0.9(1.9) 1 2 (1 9) n.d
17 (3.0) nd nd 48 (1 5) 22 (3 9)
10-
1-
0
I
7
10/tg NP-CG
B 100-
• •
o at
!
D
•
10-
1-
B
B
• •D
a
a
D
a
• •
a
0.10
1 2
4
6
weeks of anti-CD4 treatment Fig. 4. Chronic anti-CD4 treatment impairs the ability to mount an adoptive transfer response. Groups of mice received 2 x 107 spleen cells from nonpnmed mice (A) or from mice primed with NP-CG and later treated for the indicated lengths of time with anti-CD4 ( • , • ) Closed symbols' pnming with alum-precipitated antigen plus B.pertussis. Open symbols, pnming with alum-precipitated antigen only. The figure shows the trters of antigen-specific antibodies in the serum of individual mice on day 12 after boosting
spleen cells from the mice that had been primed and treated as described in the previous experiment. Since under the conditions of that experiment B cells from anti-CD4-treated and control animals produce indistinguishable adoptive responses when sufficient T cell help is provided (Table 2), any impairment in the response obtained from total spleen cells would be due to lack of T cell help The result of this experiment is shown in Fig. 4, where the titers from individual mice are plotted. Clearly, helper
14
30 days
Fig. 5. Anti-CD4 treatment inhibits the in situ secondary response Mice were immunized on day 0 with 10 /ig NP - CG without adjuvant. Serum trters of napten-specrtlc lgG1 antibodies in the serum before immunization and on days 7, 14, and 30 are shown • , unpnmed mice; O, mice that had been primed with NP-CG 10 weeks previously, • , mice that had been primed with NP-CG 10 weeks before and then were injected with anti-CD4 antibodies for 1 week before immunization Six mice in each group activity can be detected in the adoptive secondary response after anti-CD4 treatment of the donors. Some loss of help seems to occur with time, as longer periods of treatment result in somewhat lower adoptive responses spread over a wide range, with some mice failing to respond at all. Taken together, the results of this experiment indicate that anti-CD4 treatment does not completely eliminate CD4-positive T cells from the animal. Possibly the C D 4 - C D 8 - T cells present in the spleen in the early period of treatment (Fig. 1) belong to the CD4 lineage, and after transfer into nontreated recipients are able to expand and/or re-express the CD4 molecule and thus help the memory B cells present to make a response. With longer periods of treatment more and more of these cells are eliminated, resulting, after adoptive transfer, in a helper activity that is less and less efficient Because of this experimental result it became mandatory to assess helper activity in the anti-CD4-treated animals more directly. This was done by analyzing the capacity of the manipulated animals to mount a secondary response in situ, in the presence of anti-CD4 antibodies. In accord with earlier data (14,15), a secondary response could not be elicited in this situation (Fig. 5) Inhibition amounts to a factor of 10 at least; background levels of serum antibodies do not permit us to determine whether the reduction is even larger. The absence of a secondary response in these mice could be due to suppression of the antigen-specific response. This seems unlikely as after recovery from anti-CD4 treatment the mice are able to mount an anti-NP immune response (see Fig. 2). Our interpretation of these results is that in the anti-CD4-treated mice the circulating anti-CD4 antibodies block the accessory molecule (or lead to its
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n d , not determined "Priming was done with alum-precipitated antigen (experiment 1) or alum-precipitated antigen plus B.pertussis (experiment 2). Cefl transfer was performed 10 (experiment 1) or 12 weeks (experiment 2) after pnming bGeometric means with standard deviation coefficients of haptenbmding lgG1 serum antibody concentration (^g/ml) on day 12 after transfer of 107 CG-pnmed T cells together with the indicated number of B cells from NP - CG-pnmed mice which were treated with anti-CD4 for the last 1, 2, 4, or 6 weeks before transfer as indicated (mean of 3 - 5 mice/group). Serum trters of mice that received 107 CG-pnmed T cells plus either 107 B cells from nave donors or 107 B cells from primed donors but were not boosted with antigen after transfer were < 0 14 /ig/ml.
s anti-NIF
Anti-CD4 treatment (weeks)
492
Memory B cells persist under anti-CD4 treatment
3:
10-
c
1-
CD
0.1 5
8 11 14
21
28 days
modulation) in the cells that are not eliminated, thereby rendering them unable to deliver helper signals to B cells. Very few if any functional CD4+ helper cells remain under treatment. To generate a memory T cell compartment CD4+ cells are needed for the first 3 weeks after immunization If our conclusion that treatment of mice with anti-CD4 antibodies inhibits the generation of memory B cells but does not affect established memory is correct, then this system of anti-CD4 treatment should allow an estimate of the time that is necessary to generate a full memory B cell compartment after immunization. Groups of antigen-primed mice were treated with anti-CD4, starting at different times after immunization. B cells from each of these groups were taken on day 35 after priming and transferred together with carrier-specific helper cells into irradiated recipients that were then boosted with antigen. The antibody trters in the sera of the recipient mice on day 12 after boosting are shown in Fig. 6. The result shows that the groups in which treatment was started less than 21 days after immunization did not generate a full memory compartment; only after day 21 is the adoptive response indistinguishable from the control. This result is compatible with previous estimates of 2 - 4 weeks as the time necessary to generate a full memory compartment (1,34,35). It supports our conclusion that CD4 + cells are necessary to induce memory, but that whatever memory is already present is not lost upon treatment with anti-CD4 antibodies. Discussion In this work we investigated the effect of blocking helper T cell function at different stages of the immune response to a T cell-dependent antigen. Treatment of mice with anti-CD4 antibodies eliminates most CD4+ cells from peripheral lymphoid organs (Fig 1). The inhibition of the primary and secondary antibody responses (Figs
An alternative interpretation is that the circulating anti-CD4 antibodies do not block the few CD4+ cells that remain. These may represent a population of primed cells whose responses have higher affinity to antigen. This would make the interaction between primed CD4 + cells and B cells less sensitive to blocking of the accessory molecule. This interpretation is contradicted by the finding that the secondary response (where primed T cells are already present) is also inhibited by treatment with anti-CD4 (Fig. 5). If primed T cells were able to help B cells in treated mice one would not expect the secondary response to be inhibited by anti-CD4 treatment. One might thus resort to the possibility that an interaction of primed helper T with memory B cells in the phase of memory maintenance may take place in a 'protected' environment where the concentration of CD4-specific antibodies is not high enough to block the interaction of the CD4 molecule with its target or to eliminate the helper cells altogether The thymus may represent such an environment (16), but is hardly the site of B cell memory maintenance. Another microenvironment which could be considered in this context would be the germinal centers which, like the thymus, represent dense lymphocyte accumulations. However, germinal centers form in the phase of B cell memory induction (which is inhibitable by anti-CD4 antibodies) and disappear thereafter (38). Thus, the simplest interpretation of the present data is that antigen priming results in the generation of memory B cells that are long-lived and persist in the organism, able to mount a secondary antibody response if helper T cells are available. The late primary antibody response (and the secondary response) would be produced by long-lived plasma cells generated after immunization, in parallel with the generation of memory B cells Long-lived, IgG-secreting plasma cells exist in the bone marrow (39,40). It has also been found that plasma cells which migrate to the bone marrow appear late in the primary response and in secondary responses (41,42). If this interpretation is correct, one would predict that memory B cells are also antigen-independent, requiring antigen only for the initiation of the response. Evidence to the contrary obtained in mice (1,4), and recently reproduced in rats (43), could be the
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Fig. 6. Anti-CD4 treatment interrupts the build up of B cell memory. Groups of mice received 107 cells from CG-primed mice together with 107 B cells from. A , unpnmed mice, • , mice that were primed with antigen and then chronically treated with anti-CD4 starting on the indicated days after immunization, • , antigen-pnmed nontreated mice All mice were boosted 1 day after cell transfer and their serum responses measured on day 12 after boost. NIP+71 + responses are shown (5 mice/group)
2 and 5) and of the induction of B cell memory (Table 1) shows that CD4+ cells are required for these processes However, this is true only for the early phase after immunization. When treatment with anti-CD4 is delayed until 3 weeks or more after immunization little or no effect is observed: the ongoing antibody response is unaffected (Fig. 3) and there is no detectable loss of B cell memory (Table 2). We interpret these results as suggesting that the maintenance of both an ongoing antibody response and of memory in the B cell compartment are helper T cell independent. This interpretation rests on the assumption that anti-CD4 treatment effectively blocks all helper T cell-dependent mechanisms, preventing the few CD4+ cells remaining under treatment from delivering help to B cells. Treatment with anti-CD4 antibodies may inhibit helper T cell-dependent functions not only by eliminating most CD4+ cells but also by leading to the modulation of the accessory molecule and directly blocking the cells that remain. All of this would result in the complete inhibition of CD4+ T cell - B cell interactions. Supporting this view is the finding that treatment of mice with Fab fragments of CD4-specific antibodies fully inhibits the response to T cell-dependent antigens with minimal depletion of CD4+ cells (36,37).
Memory B cells persist under anti-CD4 treatment
Whatever the mechanism is that leads to the maintenance of memory in the B cell compartment, the fact that the amount of help (if any) needed to maintain memory B cells is less than that necessary to stimulate naive B cells and drive them into the memory pathway argues against the idea that a continuous recruitment of naive B cells (11) is the major mechanism of perpetuation of immunological memory to T celldependent antigens.
Acknowledgements We are grateful to Irmgard Forster for discussion, advice, and help with the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft through SFBs 74 and 243, and by the Fazrt Foundation. Abbreviations CG ELISA NP
chicken -^-globulin enzyme-linked immunosorbent assay (4-hydroxy-3-nrtropheny()acetyl
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result of the cell transfer system used, and does not directly address the question of antigen dependency of memory B cells because the loss of memory responses observed after transfer of cells into irradiated, non-primed animals could be due to lack of memory T cells or to cellular competition in the repopulation of the host's immune system. Since at this point one cannot totally exclude that in antigenprimed, anti-CD4-treated mice some helper T cells are functional and able to deliver help to B cells, we believe that the minimal conclusion from the experiments reported here is that different amounts of T cell help are needed at different stages of the response to T cell-dependent antigens. Early stages of immune responses to antigen are strictly helper T cell-dependent and are fully inhibited by anti-CD4 treatment; later stages are less or not at all dependent on help from CD4+ T cells. The former include the induction of the primary and secondary responses (14-18)— the functions classically used to define thymus(helper)-dependent antigens and responses (44-47). Our work demonstrates that the generation of a memory B cell compartment belongs also to this category. Events that take place after the induction of the immune response, like the persistence of memory in the B cell compartment and the maintenance of an ongoing primary antibody response, require less or no help from CD4+ cells.
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