Eur. J. Immunol. 1991.21: 1433-1438
Sally J. Meding and Jean Langhorne Max-Planck-Institutfur I mmunbiologie, Freiburg
CD4+ Tcells and B cells transfer immunity to t? chabaudi
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CD4+ T cells and B cells are necessary for the transfer of protective immunity to Phsmodium chabaudi chabaudP It is shown here that B cells, in addition to CD4+ Tcells, are necessary for the development of protective immunity to Plasmodium chabaudi chabaudi ( I ! chabaudi) in mice. Reconstitution of severe combined immunodeficient (SCID) mice with immune or normal CD4+ Tcells protected the majority of mice against an otherwise lethal challenge but the mice were unable to clear their parasitemias. By contrast, transfer of the same T cell populations into athymic nulnu mice enabled the recipients to control and clear their infections, immune CD4+ Tcells being most effective. Furthermore, SCID mice given CD4+ T cells from immune and normal donors simultaneously with immune B cells also could eliminate their infection. Clearance of parasitemia correlated with the presence of malariaspecific antibodies in the serum. The role of B cells and CD4+ T cells in the protective immune response to I! chabaudi is discussed.
1 Introduction The protective immune response to the erythrocytic stages of Plasmodium is dependent on CD4+ T cells [l-41. However, the apparent functional heterogeneity of this T cell subpopulation [5] has made it more difficult to elucidate the effector mechanisms involved in the clearance of infection. Currently, a concept is favored that antibody-independent mechanisms are of great importance, at least in mouse model infections with Plasmodium chabaudi and I! vinckei. This is based on earlier studies of Grun and Weidanz [6,7] and more recent experiments of Cavacini et al. [8] and Kumar et al. [4], who have shown that mice depleted of B cells are able to control their infections often to subpatent levels. Furthermore, the ability of passively transferred T cells, CD4+ T cells and T cell lines, but not immune sera, to convey protection would appear to confirm these findings [ l , 2, 4, 9, 101. It is supposed that parasites are destroyed by products of MQ, activated via T cell cytokines. However, in most of these transfer studies athymic nulnu, irradiated or CD4+ T cell-depleted recipients were used. Therefore, the presence of B cells and generation of specific antibodies cannot be ruled out.
at a time when malaria-specific IgG antibodies were present in the plasma, had little effect on the ability of mice to control their infections [12]. This evidence would suggest that at least in I! chabaudi infections, mechanisms dependent on B cells and antibodies may be important in the final clearance of the parasites from the blood and the maintenance of immunity. To clarify this, an adoptive transfer system is needed in which the presence of B cells and antibodies can be controlled. We have used the severe combined immunodeficient (SCID) mouse as recipient in cell transfer experiments. SCID mice are lacking both Tand B cells [13] and generally succumb to an infection of R chabaudi.The donor CD4+ T cells were obtained from mice undergoing a primary infection with R chabaudi which have a predominant phenotype characteristic of inflammatory-type or Th 1 cells [ 5 ] , from immune mice with a dominant B cell-helper or Th2 phenotype [ 5 , 14, 151 or from normal mice. The ability of these cells to confer protective immunity in nulnu mice has been compared with that in SCID mice in the presence and absence of simultaneously transferred immune B cells.
2 Materials and methods By contrast, we have found little evidence that infections with I! chabaudi chabaudi ( R chabaudi) are controlled by effector mechanisms mediated by CD4+ T cell-derived IFN-y [ l l ] . Furthermore, in vivo depletion of CD4+ T cells,
[I 92681
* This work was supported by the UNDPlWorld BanWWHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Rockefeller Foundation. Correspondence: Jean Langhorne, Max-Planck-Institut fur
Immunbiologie, Postfach 1169, D-7800 Freiburg, FRG Abbreviations: SCID: Severe combined immunodeficient (mouse) I? chabaudi: Plasmodium chabaudi chabaudi 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
2.1 Mice Male or female C.B-17 SCID mice (H-2d, Ighb) and female BALBlc (H-2d, Igha) mice aged 8 to 14 weeks were bred and maintained under specific pathogen-free (SPF) conditions in our own breeding facilities. BALBlc nulnu mice aged 8-10 weeks were obtained from Bomholtgdrd (Ry, Denmark). 2.2 Parasites R chabaudi chabaudi (AS), a gift of Dr. K. N. Brown, National Institute for Medical Research, London, GB,were maintained as an erythrocytic infection in BALBlc mice by 0014-298019110606-1433$3.50+ .25/0
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Eur. J. Immunol. 1991. 21: 1433-1438
S. J. Meding and J. Langhorne
injection of 1 x lo4 infected erythrocytes. Parasitemias were monitored by examination of Giemsa-stained blood films.
2.3 Antibodies and preparation of cells
T cells were prepared from spleens (10-25 spleens) by passing over nylon wool columns as described [16]. CD4+
cn
-
T cells were obtained by negative selection using anti-Ly-2 mAb (YTS 169.2 [ 171) and goat anti-rat Ig-coupled magnetic beads (Paesel, Frankfurt, FRG) to remove CD8+ cells. B lymphocyte-enriched populations were obtained from spleens by removal of T cells using mAb specific for Thy-1.2 (Jlj, [MI) and Ly-1 (C3P0, [19]) as described previously [20]. Purity of the separated cell populations was assessed by FCM (see Sect. 2.4).
2.4 FCM
100 a
0
Fluoresceinated (FL) anti-Thy-1.2, anti-Ly-2, PE-labeled anti-L3T4 and anti-mouse Ig mAb were obtained from Becton Dickinson (Mountain View, CA). FL-goat antimouse Ig and FL-goat anti-rat Ig were obtained from Jackson Immunoresearch Inc. (Bar Harbor, ME). mAb 14.8 [21], RA33A1 and RA36B2 [22], specific for the high-molecular weight component of the T200 molecule (B220), were used to detect B lymphocytes. FcR binding of some of these antibodies was prevented by addition of anti-mouse FcR mAb (2.462, [23]). Staining of lymphocytes was carried out as described previously [24] and FCM analyses were performed on a FACScan (Becton Dickinson) using the FACScan software.
Y
10 1
0.1
-9
7
Q
0.01
C Q
2 Q
R
a
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Ill
c I-
2.5 Malaria-specific ELISA assays and immunoprecipitation
10000 7
1000 1
0
I
I
I
I
10
20 TIME (days)
30
40
Figure 1. (A) Courses of infection of P chabaudi in BALBk nulnu female mice reconstituted with CD4+ cells. Transfer of 2 x lo7 normal CD4+ Tcells (W-W). CD4+ T cells from early in infection (0-0) and from late in infection (A-A). The infection in unreconstituted nulnu mice (0-0) and control euthymic BALBk (O--O) are also shown. The values represent the geometric means of the parasitemias of three to five mice. SEM were calculated to be 15% or less of the mean values. (B) I! chabaudi-specific IgG antibodies detected in the plasma of BALBk nulnu mice reconstituted with CD4+ T cells. Symbols are as above. Unreconstituted nulnu mice are represented by (+---+).
Malaria-specific IgG antibodies were measured at various intervals during infection with an ELISA assay using a lysate of the erythrocytic stages of P chabaudi as described [25]. Alkaline phosphatase-goat anti-mouse IgG was obtained from Southern Biotechnology (Birmingham, AL). SDS-PAGE and immunoprecipitation using [35S]methionine (Amersham, Braunschweig, FRG)-labeled extracts of f? chabaudi were performed as described [25].
2.6 Adoptive transfer experiments Female SCID (C.B-17) mice were first tested for the presence of Ig in the plasma.Those mice with < 250 ngof Ig were used for experiments. Separated immune or normal CD4+-enriched T cells (2 x 107/mouse)in the presence or absence of 1.5 x lo7immune B cells were injected by the i.v. route into SCID or BALB/c nulnu mice.
Table 1. FCM analysis of spleen cells from BALBk nulnu mice reconstituted with CD4+ Tcells
Donor cells used for transfer Normal Early.) Late') Unreconstituted nulnu Intact infected BALB/c
Total no. of celldspleen
Percentage of cells staining
x10P
CD4+
CDS+
B220+
428
12.9
20 I 380
8.0
0.5 0.6
75.5 73.4 66.2
8.7 1.4
0.9
533
0.6
13.1
31 1
21.9
4.5
61.2
a) Early and late denote CD4+ Tcells obtained from the spleens of mice which have had an infection with P chabaudi for 7 and 30 days, respectively.
Eur. J. Immunol. 1991.21: 1433-1438
CD4+ Tcells and B cells transfer immunity to F! chabaudi
3 Results 3.1 Transfer of CD4+ T cells in BALBk nulnu recipients
Splenic CD4+ T cells were prepared from either naive BALBlc mice or mice which had been infected with 10“ I? chabaudi parasites 7 days (Early) and 30 to 40 days (Late) previously. The purity of the subpopulations as analyzed by FCM was as follows: 70%-85% CD4+ cells,
0.01
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0.1%-2% CD8+ cells, 1%-2% B cells and 15%-30% “Null” cells. Recipient nulnu mice were injected i.v. with 2 x lo7cells of the enriched CD4+ Tpopulations and on the following day infected with 104 I? chabaudi organisms.The course of infection in the reconstituted mice is shown in Fig. 1A. Unreconstituted nulnu mice had a peak parasitemia of approximately 40% which was reduced to 10%-25% but never cleared. Of these mice 50% died within 40days. Mice reconstituted with early, late and normal CD4+ Tcells all developed an ascending parasitemia with kinetics similar to normal euthymic BALBk mice. However, all reconstituted mice were able to control and clear the infection to subpatent levels. A second wave of parasitemia was generally observed before clearance. After 35 days the various groups of mice were killed and the reconstitution with CD4+ T cells of the spleens was examined by FCM (Table l).The high total number of cells recovered from spleens in some of the groups reflects the splenomegaly seen in this infection. In general, this regresses upon clearance of parasitemia. In all cases CD4+ T cells could be recovered in numbers corresponding to 30%-50% of normal euthymic mice.The numbers of CD8+ T cells were low and not significantly different from those of unreconstituted nulnu mice. Similarly, the numbers of B cells as determined by surface Ig (not shown) and the presence of the B cell-specificsurface molecule, B220, were similar in all reconstituted groups.
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0.001
a
Malaria-specific IgG antibodies, as determined by an ELISA assay, were present in all groups of reconstituted mice, with titers comparable to euthymic control mice at the same time of infection (Fig. 1B). Unreconstituted nulnu mice had negligible levels of malaria-specific IgG antibodies (10- to 40-fold less than the reconstituted mice). 3.2 Transfer of CD4+ T cells into SCID mice in the presence and absence of immune B cells
The finding that nulnu mice reconstituted with CD4+ T cells were able to control an infection of P chabaudi does not elucidate whether these CD4+ Tcells can effect their 0.1 7 protective capacity without B cells or antibodies. Therefore, the same adoptive transfer experiments were per0.01 7 formed in SCID mice lacking bothTand B cells (Fig. 2A). Mice reconstituted with CD4+ T cells taken from mice 0.001 . , , , , , I , early or late in infection or from normal mice were unable 1 0 5 10 15 20 25 30 to clear their parasitemias. The individual courses of TIME (days) infection were similar to those of unreconstituted SCID mice for the first 15 to 20 days. After this time all Figure 2. Courses of infection of F! chabaudi in reconstituted SCID mice. (A) Reconstitution with CD4+ T cells. Transfer of unreconstituted SCID mice died from infection. By con2 x lo7 CD4+ Tcells from normal mice (W-W) from early in trast, more than 70% of all of the CD4+ Tcell-reconstituted infection (0-0) and from late during infection (A-A). The mice survived for the duration of the experiment indicating that Tcell transfer resulted in partial protection but not in course of infection of control BALB/c mice is shown by (0-0) and of unreconstituted SCID mice by (0-0). (B) Reconstitution clearance of parasitemia.The inability to transfer immunity with CD4+T cells from late in infection where antibodies were not was not due to a lack of reconstitution of these mice, since and where FCM analyses showed substantial numbers of CD4+ Tcells present in the plasma of the recipient mice (A-A) malaria-specificantibodies were detectable (0-0). Other sym- in the spleens (Table 2, Normal, Early and Late 1). As is bols are as in (A) above. (C) Reconstitution with CD4+ Tcells in the case with infected nulnu mice, all SCID mice had the presence of 1.5 x lo7 immune B cells. Symbols are as in (A). pronounced splenomegaly during infection. Mice reconstituted with immune B cells only are shown (M).
1
u
I
The parasitemias are the geometric means of eight to ten mice and SEM were calculated to be 10% or less of the mean values obtained.
Despite the presence of low numbers of B cells in these mice, no significant malaria-specific IgG antibodies could
Eur. J. Immunol. 1991. 21: 1433-1438
S. J. Meding and J. Langhorne
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Table 2. FCM analysis of spleen cells from SCID mice reconstituted with CD4+ Tcells
Donor cclls uscd for transfer
Total no. o f ccllslsplcen x 10F
Normal Early"' Late 1;'' Late 2il.h) Unreconstituted SCID Intact infected BALBk
Pcrcentaee of cells staining Y
CD4+
CD8+
B220'
I070 I720 1332
11.2 5.8
52 3YX
28.9
n.oh 1 .n 0.2 2.1
0.5 8.7 11.0 20.3
720
25.6
0.2 7.0
36.7
7.1 0.08
be detected (Fig. 3A). Antibody titers remained the same as in infected reconstituted SCID mice in the majority of CD4+ Tcell-reconstituted mice. In two instances, however, mice reconstituted with late CD4+ Tcells were able to clear their infection (Fig. 2B). Significant levels of malariaspecific IgG antibodies (Fig. 3A) and a greater proportion of B220+ cells were observed in the spleens of these mice (Table 2, Late 2) suggesting that contaminating malariaspecific B cells were present in the transferred population. These data indicated that parasite clearance in CD4+ T cell-reconstituted SCID mice only occurs in the presence of malaria-specific B cells and their respective antibodies. To confirm this finding, adoptive transfer experiments with
looooo
3
0.2
a) SeeTable 1. b) Late 2 denotes mice reconstituted with late CD4+ T cells which had detectable malaria-specific antibodies and cleared their parasitemias. Accordingly splenomegaly was also reduced.
CD4+ T cells were repeated in the presence of immune B cells. Immune B cells (1.5 x 107/mouse) prepared from mice which had received an infection 1 to 2 months previously and containing < 8% T cells (data not shown) were co-transferred into SCID mice with 2 x lo7 early, late or normal CD4+ T cells. When immune B cells were co-transferred with early and late CD4+ T cells, mice cleared their parasitemias to < 0.01% within 20 days (Fig. 2C). The courses of infection were similar to those obtained in infected euthymic mice. Reconstitution with normal CD4+ Tcells and immune B cells resulted in a slower clearance of parasites. Unreconstituted SCID mice, as in the previous experiments were unable to clear their infections and died within 20 to 30 days. FCM analysis of spleens and LN of the CD4+ T cell- and B cell-reconstituted mice showed levels of CD4+ T cells similar to those of the previous experiments and substantial numbers of B cells (data not shown). Most importantly, these mice had malaria-specific IgG antibodies (Fig. 3B) with titers greater than or similar to euthymic mice at the same times of infection. Mice reconstituted only with B cells also developed IgG antibodies of lower titers and survived but did not clear their infections.
U
w
tI-
z
PF f
-
MW 100000
1
1
2
3
4
5
6
7
8
9
200-
looo04
926946-
0
10
20
30
40
TIME (days)
Figure 3. Malaria-specific IgG antibodies detected in the plasma of SCID mice reconstituted with normal CD4+ Tcells. (A) SCID mice reconstituted with CD4+ T cells only. Transfer of 2 X 10' normal CD4+ T cells (B-H). with CD4+ T cells from early (0-0) and from late (A-A) during infection. The antibody titer of plasma from intact infected BALBlc mice is also shown (0--0)as is that of mice reconstituted with late CD4+ Tcells which cleared their infections (V-V). (B) SCID mice reconstituted with CD4+ Tcells and 1.5 x 10' immune B cells. Symbols are as in (A) above. The titers in the plasma of SCID mice receiving These titers are the B cclls only are also shown (M). eeometric means from three to five individual mice. SEM were v 20% or less.
Figure 4. SDS-PAGE of [3sS]methionine-labeled extracts of the erythrocytic stages of F! chabaudi (A) immunoprecipitated on protein A-Sepharose with pooled plasma samples taken from three to five reconstituted SCID mice. Lane 1 14C-labeled molecular weight markers; lane 2: BALBlc immune serum: lane 3: mice reconstituted with CD4+ Tcells only,which cleared their infections: lanes 4 and 5 : mice reconstituted with early and late CD4+ Tcells. respectively, together with immune B cells; lane 6: unreconstituted SCID mice; lanes 7-9: mice reconstituted with normal. early and late CD4+ Tcells. respectively, without B cells.
Eur. J. Immunol. 1991. 21: 1433-1438
CD4+ Tcells and B cells transfer immunity to fl chahaudi
Immunoprecipitation studies with [35S]methionine-labeled I? chabaudi were performed with sera obtained from the different groups of reconstituted mice (Fig. 4). Sera of mice reconstituted with CD4+ T cells and immune B cells contained antibodies with specificities for many plasmodia1 antigens similar to those of euthymic mice. By contrast, sera of mice reconstituted with Tcells only contained few malaria-specific antibodies.
4 Discussion In this report, we have investigated the capacities of various CD4+ T lymphocyte subpopulations to transfer immunity against I? chabaudi into nulnu and into SCID mice. Splenic CD4+ Tcells were prepared from normal mice and from parasite-infected mice early and late in infection and were transferred into recipient SCID mice lacking both T and B cells.Whereas unreconstituted SCID mice did not survive a challenge infection, transfer of CD4+ Tcells enabled the majority of SCID mice to survive the infection. Immune CD4+ Tcells were more effective in this regard than naive CD4+ Tcells. CD4+ Tcells alone, however, did not enable SCID mice to clear their infections. By contrast, transfer of the same numbers of CD4+ T cells, obtained from early and late in infection and from normal mice, into nulnu mice resulted in the clearance of parasitemia. Accordingly, transfer of CD4+ T cells together with immune B cells enabled SCID mice to clear their infections to subpatent levels. These data extend earlier adoptive transfer experiments which showed, using irradiated mice and nulnu mice as recipients, that unfractionated spleen cells were most effective in transferring immunity to malaria infections in mice [9, 261. These results are basically in agreement with studies from our laboratory and support our previous hypothesis that immunity in mice to P chabaudi is established as a two-step process [12, 14, 151. In normal mice, the first phase of infection is characterized by a rapid increase followed by a rapid decrease in parasitemia during the first 10 days of infection. During this period, the activated CD4+ T cells appear to be of T h l cell type and few IgG antibodies are produced [14, 151. The second phase of infection after approximately 18days is characterized by a low level parasitemia with several small recrudescences and a final total parasite clearance after several weeks. During this second phase of infection, Th2-type CD4+ cells are activated and specific IgG antibodies are present [14, 151. SCID mice reconstituted with CD4+ cells without B cells appear to show the perpetuated first phase of infection with only a slight reduction in parasitemia, whereas SCID mice reconstituted with CD4+ Tcells plus B cells establish the full immunity comparable with that of normal mice. Experiments to define the role of Tcells and B cells in anti-malarial immunity have also been performed by Grun and Weidanz [6, 71, Kumar et al. [4] and Cavacini et al. [8]. These authors demonstrated that p-suppressed mice could control blood-stage infections but did not eliminate parasites completely. The difference between these results and our data lies primarily in the efficiency of the control of parasitemia in the two types of B cell-deficient mice. Whereas only few parasites survive in y-suppressed mice, CD4+ reconstituted-SCID mice without B cells show a
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much larger remaining parasitemia. Nevertheless, we feel that the discrepancies between the results obtained in y-suppressed mice and in CD4+ reconstituted SCID mice may be quantitative in nature. Most probably these differences are due to the different physiological conditions expected to exist in vivo between the two experimental models. A major difference in the models is that the y-suppressed mouse has a complete T cell compartment, whereas the SCID mouse is reconstituted with only 2 X lo7 CD4+ Tcells. It is quite possible that the larger number of cells is sufficient to control the parasitemia at lower levels. A further, if less likely, possibility is that p-suppressed mice are not antibody free; there are several milligrams of goat or rabbit immunoglobulins, which in the absence of other antibodies of higher avidity may be able to bind and eliminate erythrocytic parasites.
In addition to a difference in the numbers of effective CD4+ T cells, there may also be differences in the functional capacities of T lymphocytes. It has been shown that IFN-y, produced by Thl-type CD4+ T cells, has an inhibitory effect on both the production and the function of IL4, produced by Th2cells [27]. Conversely,Th:! cells and B cells have been shown to be effective producers of IL 10, a cytokine which inhibits IFN-y productin by Thl cells [28-301. Therefore, an initial T h l response will delay the subsequent Th2/B cell response. ATh2/B cell response, once initiated, will in turn exert a negative feedback on the Thl-type response. It is, therefore, possible that, in the absence of B cells, aThl-type response may be more effective and may continue longer than in their absence. In this regard, there is evidence in p-suppressed mice that Th function, a characteristic of Th2-type T cells, is substantially reduced compared with untreated mice [31]. This retention of a Thl-like function may be more pronounced in p-suppressed mice than in CD4+-reconstituted SCID mice since, in the former,T cells have undergone their entire development with a deficit of B cells. Accordingly, in the presence of B cells activated through Th2 cells, the Thl-mediated control of parasitemia may become less effective and the B cell- and antibodymediated parasite clearance would become the dominant mechanism of immunity.The results of Grun and Weidanz [6,7], Cavacini et al. [S] and ourselves agree in showing that full parasite clearance can only be achieved in the presence of B cells or antibody. Simple neutralization of Plasmodia by circulating antibody is probably insufficient for complete parasite clearance. As shown before, immunity cannot reliably be transferred with immune sera alone ([l, 91 and J. Langhorne, unpublished observations). Furthermore, an intact and activated spleen has been shown to be necessary for the clearance of the erythrocytic stages of Plasmodia in mice [4, 321. It is, therefore, likely that antigen-focusing and -presenting properties of B cells have profound influences on the establishment of anti-malarial immunity and that antibodydependent cellular cytotoxicity may be a major effector mechanism in parasite clearance. The authors would like to thank Klaus Eichmannfor all his helpful advice and discussions, and Dieter Hartz, Martin Goodier, Arno
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S. J. Meding and J. Langhorne
Mullbacher und Markus M . Simon for critical reading of the manuscript. The skilled technical assistance of Christiane Wirbelauer and Birgit Simon-Haarhaus is much appreciated. We thank also Uwe Meding for his help with the computer graphics and Gabi Prosch and Rose Brugger for expert editorial assistance. Received February 1. 1991.
5 References 1 Cavacini, L. A., Long. C. A. and Weidanz, W. P., Infect. Immun. 1986. 52: 637. 2 Brake, D. A., Weidanz. W. F? and Long, C. A , , J. Immunol. 1986. 137: 347. 3 Suss, G., Eichmann, K., Kury, E.. Linke, A. and Langhorne, J., Infect. Immun. 1988. 56: 3081. 4 Kumar, S., Good, M. F., Dontfraid, F.,Vinetz, J. M. and Miller, L. H., J. Immunol. 1989. 143: 2017. 5 M0smann.T. R., Cherwinski, H., Bond, M.W., Giedlin, M. A. and Coffman. R. L.. J. Immunol. 1986. 136: 2348. 6 Grun, J. L. and Weidanz. W. I?, Nature 1981. 290: 143. 7 Grun, J. L. and Weidanz, W. P., Infect. Immun. 1983. 41: 1197. 8 Cavacini, L. A., Parke, L. A. and Weidanz, W. I?, Infect. Immun. 1990. 58: 2946. 9 McDonald, V. and Phillips. R. S.. Immunology 1978. 34: 821. 10 Brake, D. A., Long, C. A. and Weidanz, W. P., J. Immunol. 1988. 140: 1989. 11 Meding, S., Cheng, S. C., Simon-Haarhaus, B. and Langhorne, J., Infect. Immun. 1990. 58: 3671. 12 Langhorne, J.. Simon-Harhaus, B. and Meding, S.J., Immunol. Lett. 1990. 25: 101. 13 Bosma, G. C., Custer, R. I? and Bosma, M. J., Nature 1983.301: 527.
Eur. J. Immunol. 1991. 21: 1433-1438 14 Langhorne, J., Gillard, S., Simon. B., Slade, S. and Eichmann. K., Int. Immunol. 1989. I : 416. 15 Langhorne, J., Meding, S. J., Eichmann, K. and Gillard, S.. Immunol. Rev. 1989. 112: 71. 16 Julius, M. H., Simpson, E. and Herzenberg, L. A , , Eur. J. Immunol. 1973. 3: 645. 17 Cobbold, S. I?, Jayasuriya, A., Nash, A., Prospero,T. D. and Waldmann, H., Nature 1984. 312: 548. 18 Bruce, J., Symington. F. W., McKearn, T. J. and Sprent, J.. J. Imrnunol. 1981. 127: 2496. 19 Ledbetter, J. A. and Herzenberg, L. A., Immunol. Rev. 1979. 47: 63. 20 Langhorne. J. and Simon, B., Parasit. Immunol. 1989. 11: 545. 21 Kincade, I? W., Lee, G., Watanabe, T., Sun, L. and Scheid, M.P., J. Immunol. 1981. 127: 2262. 22 Coffman, R. L., Immunol. Rev. 1982. 69: 5. 23 Unkeless, J. C., J. Exp. Med. 1979. 150: 580. 24 Langhorne, J. and Titus, J., Eur. J. Immunol. 1988. 18: 1. 25 Langhorne, J., Evans, C. B., Asofsky, R. and Taylor, D. W., Cell. Immunol. 1984. 87: 452. 26 Brinkmann,V., Kaufmann, S. H. E. and Simon, M. M., Infect. Immun. 1985. 47: 737. 27 Snapper, C. M. and Paul, W. E., Science 1987. 236: 944. 28 Horowitz, J. B., Kay, J., Conrad, P. J., Katz, M. E. and Janeway, C. A,, Proc. Natl. Acad. Sci. USA 1986. 83: 1886. 29 Fiorentino, D. F., Bond, M. W. and Mosmann,T. R., J. Exp. Med. 1989. 170: 2081. 30 OGarra, A., Stapleton. G., Dhar,V., Pearce, M., Schumacher, J., Rugo, H., Barbis, B., Stall,A., Cupp, J., Moore, K.,Vieira, €?, Mosmann,T. R.,Whitmore, A., Arnold, L., Haughton, G. and Howard, M., Infect. Immun. 1990. 2: 821. 31 Kim, K. J., Rollwagen, F., Asofsky. R. and Lefkovits, I.. Eur. J. Immunol. 1984. 14: 476. 32 Grun, J. L., Long, C. A. and Weidanz,W. I?, Infect. Immun. 1985. 48: 853.
Sally J. Meding and Jean Langhorne Max-Planck-Institutfur I mmunbiologie, Freiburg
CD4+ Tcells and B cells transfer immunity to t? chabaudi
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CD4+ T cells and B cells are necessary for the transfer of protective immunity to Phsmodium chabaudi chabaudP It is shown here that B cells, in addition to CD4+ Tcells, are necessary for the development of protective immunity to Plasmodium chabaudi chabaudi ( I ! chabaudi) in mice. Reconstitution of severe combined immunodeficient (SCID) mice with immune or normal CD4+ Tcells protected the majority of mice against an otherwise lethal challenge but the mice were unable to clear their parasitemias. By contrast, transfer of the same T cell populations into athymic nulnu mice enabled the recipients to control and clear their infections, immune CD4+ Tcells being most effective. Furthermore, SCID mice given CD4+ T cells from immune and normal donors simultaneously with immune B cells also could eliminate their infection. Clearance of parasitemia correlated with the presence of malariaspecific antibodies in the serum. The role of B cells and CD4+ T cells in the protective immune response to I! chabaudi is discussed.
1 Introduction The protective immune response to the erythrocytic stages of Plasmodium is dependent on CD4+ T cells [l-41. However, the apparent functional heterogeneity of this T cell subpopulation [5] has made it more difficult to elucidate the effector mechanisms involved in the clearance of infection. Currently, a concept is favored that antibody-independent mechanisms are of great importance, at least in mouse model infections with Plasmodium chabaudi and I! vinckei. This is based on earlier studies of Grun and Weidanz [6,7] and more recent experiments of Cavacini et al. [8] and Kumar et al. [4], who have shown that mice depleted of B cells are able to control their infections often to subpatent levels. Furthermore, the ability of passively transferred T cells, CD4+ T cells and T cell lines, but not immune sera, to convey protection would appear to confirm these findings [ l , 2, 4, 9, 101. It is supposed that parasites are destroyed by products of MQ, activated via T cell cytokines. However, in most of these transfer studies athymic nulnu, irradiated or CD4+ T cell-depleted recipients were used. Therefore, the presence of B cells and generation of specific antibodies cannot be ruled out.
at a time when malaria-specific IgG antibodies were present in the plasma, had little effect on the ability of mice to control their infections [12]. This evidence would suggest that at least in I! chabaudi infections, mechanisms dependent on B cells and antibodies may be important in the final clearance of the parasites from the blood and the maintenance of immunity. To clarify this, an adoptive transfer system is needed in which the presence of B cells and antibodies can be controlled. We have used the severe combined immunodeficient (SCID) mouse as recipient in cell transfer experiments. SCID mice are lacking both Tand B cells [13] and generally succumb to an infection of R chabaudi.The donor CD4+ T cells were obtained from mice undergoing a primary infection with R chabaudi which have a predominant phenotype characteristic of inflammatory-type or Th 1 cells [ 5 ] , from immune mice with a dominant B cell-helper or Th2 phenotype [ 5 , 14, 151 or from normal mice. The ability of these cells to confer protective immunity in nulnu mice has been compared with that in SCID mice in the presence and absence of simultaneously transferred immune B cells.
2 Materials and methods By contrast, we have found little evidence that infections with I! chabaudi chabaudi ( R chabaudi) are controlled by effector mechanisms mediated by CD4+ T cell-derived IFN-y [ l l ] . Furthermore, in vivo depletion of CD4+ T cells,
[I 92681
* This work was supported by the UNDPlWorld BanWWHO Special Programme for Research and Training in Tropical Diseases (TDR) and the Rockefeller Foundation. Correspondence: Jean Langhorne, Max-Planck-Institut fur
Immunbiologie, Postfach 1169, D-7800 Freiburg, FRG Abbreviations: SCID: Severe combined immunodeficient (mouse) I? chabaudi: Plasmodium chabaudi chabaudi 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991
2.1 Mice Male or female C.B-17 SCID mice (H-2d, Ighb) and female BALBlc (H-2d, Igha) mice aged 8 to 14 weeks were bred and maintained under specific pathogen-free (SPF) conditions in our own breeding facilities. BALBlc nulnu mice aged 8-10 weeks were obtained from Bomholtgdrd (Ry, Denmark). 2.2 Parasites R chabaudi chabaudi (AS), a gift of Dr. K. N. Brown, National Institute for Medical Research, London, GB,were maintained as an erythrocytic infection in BALBlc mice by 0014-298019110606-1433$3.50+ .25/0
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Eur. J. Immunol. 1991. 21: 1433-1438
S. J. Meding and J. Langhorne
injection of 1 x lo4 infected erythrocytes. Parasitemias were monitored by examination of Giemsa-stained blood films.
2.3 Antibodies and preparation of cells
T cells were prepared from spleens (10-25 spleens) by passing over nylon wool columns as described [16]. CD4+
cn
-
T cells were obtained by negative selection using anti-Ly-2 mAb (YTS 169.2 [ 171) and goat anti-rat Ig-coupled magnetic beads (Paesel, Frankfurt, FRG) to remove CD8+ cells. B lymphocyte-enriched populations were obtained from spleens by removal of T cells using mAb specific for Thy-1.2 (Jlj, [MI) and Ly-1 (C3P0, [19]) as described previously [20]. Purity of the separated cell populations was assessed by FCM (see Sect. 2.4).
2.4 FCM
100 a
0
Fluoresceinated (FL) anti-Thy-1.2, anti-Ly-2, PE-labeled anti-L3T4 and anti-mouse Ig mAb were obtained from Becton Dickinson (Mountain View, CA). FL-goat antimouse Ig and FL-goat anti-rat Ig were obtained from Jackson Immunoresearch Inc. (Bar Harbor, ME). mAb 14.8 [21], RA33A1 and RA36B2 [22], specific for the high-molecular weight component of the T200 molecule (B220), were used to detect B lymphocytes. FcR binding of some of these antibodies was prevented by addition of anti-mouse FcR mAb (2.462, [23]). Staining of lymphocytes was carried out as described previously [24] and FCM analyses were performed on a FACScan (Becton Dickinson) using the FACScan software.
Y
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7
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0.01
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2 Q
R
a
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2.5 Malaria-specific ELISA assays and immunoprecipitation
10000 7
1000 1
0
I
I
I
I
10
20 TIME (days)
30
40
Figure 1. (A) Courses of infection of P chabaudi in BALBk nulnu female mice reconstituted with CD4+ cells. Transfer of 2 x lo7 normal CD4+ Tcells (W-W). CD4+ T cells from early in infection (0-0) and from late in infection (A-A). The infection in unreconstituted nulnu mice (0-0) and control euthymic BALBk (O--O) are also shown. The values represent the geometric means of the parasitemias of three to five mice. SEM were calculated to be 15% or less of the mean values. (B) I! chabaudi-specific IgG antibodies detected in the plasma of BALBk nulnu mice reconstituted with CD4+ T cells. Symbols are as above. Unreconstituted nulnu mice are represented by (+---+).
Malaria-specific IgG antibodies were measured at various intervals during infection with an ELISA assay using a lysate of the erythrocytic stages of P chabaudi as described [25]. Alkaline phosphatase-goat anti-mouse IgG was obtained from Southern Biotechnology (Birmingham, AL). SDS-PAGE and immunoprecipitation using [35S]methionine (Amersham, Braunschweig, FRG)-labeled extracts of f? chabaudi were performed as described [25].
2.6 Adoptive transfer experiments Female SCID (C.B-17) mice were first tested for the presence of Ig in the plasma.Those mice with < 250 ngof Ig were used for experiments. Separated immune or normal CD4+-enriched T cells (2 x 107/mouse)in the presence or absence of 1.5 x lo7immune B cells were injected by the i.v. route into SCID or BALB/c nulnu mice.
Table 1. FCM analysis of spleen cells from BALBk nulnu mice reconstituted with CD4+ Tcells
Donor cells used for transfer Normal Early.) Late') Unreconstituted nulnu Intact infected BALB/c
Total no. of celldspleen
Percentage of cells staining
x10P
CD4+
CDS+
B220+
428
12.9
20 I 380
8.0
0.5 0.6
75.5 73.4 66.2
8.7 1.4
0.9
533
0.6
13.1
31 1
21.9
4.5
61.2
a) Early and late denote CD4+ Tcells obtained from the spleens of mice which have had an infection with P chabaudi for 7 and 30 days, respectively.
Eur. J. Immunol. 1991.21: 1433-1438
CD4+ Tcells and B cells transfer immunity to F! chabaudi
3 Results 3.1 Transfer of CD4+ T cells in BALBk nulnu recipients
Splenic CD4+ T cells were prepared from either naive BALBlc mice or mice which had been infected with 10“ I? chabaudi parasites 7 days (Early) and 30 to 40 days (Late) previously. The purity of the subpopulations as analyzed by FCM was as follows: 70%-85% CD4+ cells,
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0.1%-2% CD8+ cells, 1%-2% B cells and 15%-30% “Null” cells. Recipient nulnu mice were injected i.v. with 2 x lo7cells of the enriched CD4+ Tpopulations and on the following day infected with 104 I? chabaudi organisms.The course of infection in the reconstituted mice is shown in Fig. 1A. Unreconstituted nulnu mice had a peak parasitemia of approximately 40% which was reduced to 10%-25% but never cleared. Of these mice 50% died within 40days. Mice reconstituted with early, late and normal CD4+ Tcells all developed an ascending parasitemia with kinetics similar to normal euthymic BALBk mice. However, all reconstituted mice were able to control and clear the infection to subpatent levels. A second wave of parasitemia was generally observed before clearance. After 35 days the various groups of mice were killed and the reconstitution with CD4+ T cells of the spleens was examined by FCM (Table l).The high total number of cells recovered from spleens in some of the groups reflects the splenomegaly seen in this infection. In general, this regresses upon clearance of parasitemia. In all cases CD4+ T cells could be recovered in numbers corresponding to 30%-50% of normal euthymic mice.The numbers of CD8+ T cells were low and not significantly different from those of unreconstituted nulnu mice. Similarly, the numbers of B cells as determined by surface Ig (not shown) and the presence of the B cell-specificsurface molecule, B220, were similar in all reconstituted groups.
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0.001
a
Malaria-specific IgG antibodies, as determined by an ELISA assay, were present in all groups of reconstituted mice, with titers comparable to euthymic control mice at the same time of infection (Fig. 1B). Unreconstituted nulnu mice had negligible levels of malaria-specific IgG antibodies (10- to 40-fold less than the reconstituted mice). 3.2 Transfer of CD4+ T cells into SCID mice in the presence and absence of immune B cells
The finding that nulnu mice reconstituted with CD4+ T cells were able to control an infection of P chabaudi does not elucidate whether these CD4+ Tcells can effect their 0.1 7 protective capacity without B cells or antibodies. Therefore, the same adoptive transfer experiments were per0.01 7 formed in SCID mice lacking bothTand B cells (Fig. 2A). Mice reconstituted with CD4+ T cells taken from mice 0.001 . , , , , , I , early or late in infection or from normal mice were unable 1 0 5 10 15 20 25 30 to clear their parasitemias. The individual courses of TIME (days) infection were similar to those of unreconstituted SCID mice for the first 15 to 20 days. After this time all Figure 2. Courses of infection of F! chabaudi in reconstituted SCID mice. (A) Reconstitution with CD4+ T cells. Transfer of unreconstituted SCID mice died from infection. By con2 x lo7 CD4+ Tcells from normal mice (W-W) from early in trast, more than 70% of all of the CD4+ Tcell-reconstituted infection (0-0) and from late during infection (A-A). The mice survived for the duration of the experiment indicating that Tcell transfer resulted in partial protection but not in course of infection of control BALB/c mice is shown by (0-0) and of unreconstituted SCID mice by (0-0). (B) Reconstitution clearance of parasitemia.The inability to transfer immunity with CD4+T cells from late in infection where antibodies were not was not due to a lack of reconstitution of these mice, since and where FCM analyses showed substantial numbers of CD4+ Tcells present in the plasma of the recipient mice (A-A) malaria-specificantibodies were detectable (0-0). Other sym- in the spleens (Table 2, Normal, Early and Late 1). As is bols are as in (A) above. (C) Reconstitution with CD4+ Tcells in the case with infected nulnu mice, all SCID mice had the presence of 1.5 x lo7 immune B cells. Symbols are as in (A). pronounced splenomegaly during infection. Mice reconstituted with immune B cells only are shown (M).
1
u
I
The parasitemias are the geometric means of eight to ten mice and SEM were calculated to be 10% or less of the mean values obtained.
Despite the presence of low numbers of B cells in these mice, no significant malaria-specific IgG antibodies could
Eur. J. Immunol. 1991. 21: 1433-1438
S. J. Meding and J. Langhorne
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Table 2. FCM analysis of spleen cells from SCID mice reconstituted with CD4+ Tcells
Donor cclls uscd for transfer
Total no. o f ccllslsplcen x 10F
Normal Early"' Late 1;'' Late 2il.h) Unreconstituted SCID Intact infected BALBk
Pcrcentaee of cells staining Y
CD4+
CD8+
B220'
I070 I720 1332
11.2 5.8
52 3YX
28.9
n.oh 1 .n 0.2 2.1
0.5 8.7 11.0 20.3
720
25.6
0.2 7.0
36.7
7.1 0.08
be detected (Fig. 3A). Antibody titers remained the same as in infected reconstituted SCID mice in the majority of CD4+ Tcell-reconstituted mice. In two instances, however, mice reconstituted with late CD4+ Tcells were able to clear their infection (Fig. 2B). Significant levels of malariaspecific IgG antibodies (Fig. 3A) and a greater proportion of B220+ cells were observed in the spleens of these mice (Table 2, Late 2) suggesting that contaminating malariaspecific B cells were present in the transferred population. These data indicated that parasite clearance in CD4+ T cell-reconstituted SCID mice only occurs in the presence of malaria-specific B cells and their respective antibodies. To confirm this finding, adoptive transfer experiments with
looooo
3
0.2
a) SeeTable 1. b) Late 2 denotes mice reconstituted with late CD4+ T cells which had detectable malaria-specific antibodies and cleared their parasitemias. Accordingly splenomegaly was also reduced.
CD4+ T cells were repeated in the presence of immune B cells. Immune B cells (1.5 x 107/mouse) prepared from mice which had received an infection 1 to 2 months previously and containing < 8% T cells (data not shown) were co-transferred into SCID mice with 2 x lo7 early, late or normal CD4+ T cells. When immune B cells were co-transferred with early and late CD4+ T cells, mice cleared their parasitemias to < 0.01% within 20 days (Fig. 2C). The courses of infection were similar to those obtained in infected euthymic mice. Reconstitution with normal CD4+ Tcells and immune B cells resulted in a slower clearance of parasites. Unreconstituted SCID mice, as in the previous experiments were unable to clear their infections and died within 20 to 30 days. FCM analysis of spleens and LN of the CD4+ T cell- and B cell-reconstituted mice showed levels of CD4+ T cells similar to those of the previous experiments and substantial numbers of B cells (data not shown). Most importantly, these mice had malaria-specific IgG antibodies (Fig. 3B) with titers greater than or similar to euthymic mice at the same times of infection. Mice reconstituted only with B cells also developed IgG antibodies of lower titers and survived but did not clear their infections.
U
w
tI-
z
PF f
-
MW 100000
1
1
2
3
4
5
6
7
8
9
200-
looo04
926946-
0
10
20
30
40
TIME (days)
Figure 3. Malaria-specific IgG antibodies detected in the plasma of SCID mice reconstituted with normal CD4+ Tcells. (A) SCID mice reconstituted with CD4+ T cells only. Transfer of 2 X 10' normal CD4+ T cells (B-H). with CD4+ T cells from early (0-0) and from late (A-A) during infection. The antibody titer of plasma from intact infected BALBlc mice is also shown (0--0)as is that of mice reconstituted with late CD4+ Tcells which cleared their infections (V-V). (B) SCID mice reconstituted with CD4+ Tcells and 1.5 x 10' immune B cells. Symbols are as in (A) above. The titers in the plasma of SCID mice receiving These titers are the B cclls only are also shown (M). eeometric means from three to five individual mice. SEM were v 20% or less.
Figure 4. SDS-PAGE of [3sS]methionine-labeled extracts of the erythrocytic stages of F! chabaudi (A) immunoprecipitated on protein A-Sepharose with pooled plasma samples taken from three to five reconstituted SCID mice. Lane 1 14C-labeled molecular weight markers; lane 2: BALBlc immune serum: lane 3: mice reconstituted with CD4+ Tcells only,which cleared their infections: lanes 4 and 5 : mice reconstituted with early and late CD4+ Tcells. respectively, together with immune B cells; lane 6: unreconstituted SCID mice; lanes 7-9: mice reconstituted with normal. early and late CD4+ Tcells. respectively, without B cells.
Eur. J. Immunol. 1991. 21: 1433-1438
CD4+ Tcells and B cells transfer immunity to fl chahaudi
Immunoprecipitation studies with [35S]methionine-labeled I? chabaudi were performed with sera obtained from the different groups of reconstituted mice (Fig. 4). Sera of mice reconstituted with CD4+ T cells and immune B cells contained antibodies with specificities for many plasmodia1 antigens similar to those of euthymic mice. By contrast, sera of mice reconstituted with Tcells only contained few malaria-specific antibodies.
4 Discussion In this report, we have investigated the capacities of various CD4+ T lymphocyte subpopulations to transfer immunity against I? chabaudi into nulnu and into SCID mice. Splenic CD4+ Tcells were prepared from normal mice and from parasite-infected mice early and late in infection and were transferred into recipient SCID mice lacking both T and B cells.Whereas unreconstituted SCID mice did not survive a challenge infection, transfer of CD4+ Tcells enabled the majority of SCID mice to survive the infection. Immune CD4+ Tcells were more effective in this regard than naive CD4+ Tcells. CD4+ Tcells alone, however, did not enable SCID mice to clear their infections. By contrast, transfer of the same numbers of CD4+ T cells, obtained from early and late in infection and from normal mice, into nulnu mice resulted in the clearance of parasitemia. Accordingly, transfer of CD4+ T cells together with immune B cells enabled SCID mice to clear their infections to subpatent levels. These data extend earlier adoptive transfer experiments which showed, using irradiated mice and nulnu mice as recipients, that unfractionated spleen cells were most effective in transferring immunity to malaria infections in mice [9, 261. These results are basically in agreement with studies from our laboratory and support our previous hypothesis that immunity in mice to P chabaudi is established as a two-step process [12, 14, 151. In normal mice, the first phase of infection is characterized by a rapid increase followed by a rapid decrease in parasitemia during the first 10 days of infection. During this period, the activated CD4+ T cells appear to be of T h l cell type and few IgG antibodies are produced [14, 151. The second phase of infection after approximately 18days is characterized by a low level parasitemia with several small recrudescences and a final total parasite clearance after several weeks. During this second phase of infection, Th2-type CD4+ cells are activated and specific IgG antibodies are present [14, 151. SCID mice reconstituted with CD4+ cells without B cells appear to show the perpetuated first phase of infection with only a slight reduction in parasitemia, whereas SCID mice reconstituted with CD4+ Tcells plus B cells establish the full immunity comparable with that of normal mice. Experiments to define the role of Tcells and B cells in anti-malarial immunity have also been performed by Grun and Weidanz [6, 71, Kumar et al. [4] and Cavacini et al. [8]. These authors demonstrated that p-suppressed mice could control blood-stage infections but did not eliminate parasites completely. The difference between these results and our data lies primarily in the efficiency of the control of parasitemia in the two types of B cell-deficient mice. Whereas only few parasites survive in y-suppressed mice, CD4+ reconstituted-SCID mice without B cells show a
1437
much larger remaining parasitemia. Nevertheless, we feel that the discrepancies between the results obtained in y-suppressed mice and in CD4+ reconstituted SCID mice may be quantitative in nature. Most probably these differences are due to the different physiological conditions expected to exist in vivo between the two experimental models. A major difference in the models is that the y-suppressed mouse has a complete T cell compartment, whereas the SCID mouse is reconstituted with only 2 X lo7 CD4+ Tcells. It is quite possible that the larger number of cells is sufficient to control the parasitemia at lower levels. A further, if less likely, possibility is that p-suppressed mice are not antibody free; there are several milligrams of goat or rabbit immunoglobulins, which in the absence of other antibodies of higher avidity may be able to bind and eliminate erythrocytic parasites.
In addition to a difference in the numbers of effective CD4+ T cells, there may also be differences in the functional capacities of T lymphocytes. It has been shown that IFN-y, produced by Thl-type CD4+ T cells, has an inhibitory effect on both the production and the function of IL4, produced by Th2cells [27]. Conversely,Th:! cells and B cells have been shown to be effective producers of IL 10, a cytokine which inhibits IFN-y productin by Thl cells [28-301. Therefore, an initial T h l response will delay the subsequent Th2/B cell response. ATh2/B cell response, once initiated, will in turn exert a negative feedback on the Thl-type response. It is, therefore, possible that, in the absence of B cells, aThl-type response may be more effective and may continue longer than in their absence. In this regard, there is evidence in p-suppressed mice that Th function, a characteristic of Th2-type T cells, is substantially reduced compared with untreated mice [31]. This retention of a Thl-like function may be more pronounced in p-suppressed mice than in CD4+-reconstituted SCID mice since, in the former,T cells have undergone their entire development with a deficit of B cells. Accordingly, in the presence of B cells activated through Th2 cells, the Thl-mediated control of parasitemia may become less effective and the B cell- and antibodymediated parasite clearance would become the dominant mechanism of immunity.The results of Grun and Weidanz [6,7], Cavacini et al. [S] and ourselves agree in showing that full parasite clearance can only be achieved in the presence of B cells or antibody. Simple neutralization of Plasmodia by circulating antibody is probably insufficient for complete parasite clearance. As shown before, immunity cannot reliably be transferred with immune sera alone ([l, 91 and J. Langhorne, unpublished observations). Furthermore, an intact and activated spleen has been shown to be necessary for the clearance of the erythrocytic stages of Plasmodia in mice [4, 321. It is, therefore, likely that antigen-focusing and -presenting properties of B cells have profound influences on the establishment of anti-malarial immunity and that antibodydependent cellular cytotoxicity may be a major effector mechanism in parasite clearance. The authors would like to thank Klaus Eichmannfor all his helpful advice and discussions, and Dieter Hartz, Martin Goodier, Arno
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S. J. Meding and J. Langhorne
Mullbacher und Markus M . Simon for critical reading of the manuscript. The skilled technical assistance of Christiane Wirbelauer and Birgit Simon-Haarhaus is much appreciated. We thank also Uwe Meding for his help with the computer graphics and Gabi Prosch and Rose Brugger for expert editorial assistance. Received February 1. 1991.
5 References 1 Cavacini, L. A., Long. C. A. and Weidanz, W. P., Infect. Immun. 1986. 52: 637. 2 Brake, D. A., Weidanz. W. F? and Long, C. A , , J. Immunol. 1986. 137: 347. 3 Suss, G., Eichmann, K., Kury, E.. Linke, A. and Langhorne, J., Infect. Immun. 1988. 56: 3081. 4 Kumar, S., Good, M. F., Dontfraid, F.,Vinetz, J. M. and Miller, L. H., J. Immunol. 1989. 143: 2017. 5 M0smann.T. R., Cherwinski, H., Bond, M.W., Giedlin, M. A. and Coffman. R. L.. J. Immunol. 1986. 136: 2348. 6 Grun, J. L. and Weidanz. W. I?, Nature 1981. 290: 143. 7 Grun, J. L. and Weidanz, W. P., Infect. Immun. 1983. 41: 1197. 8 Cavacini, L. A., Parke, L. A. and Weidanz, W. I?, Infect. Immun. 1990. 58: 2946. 9 McDonald, V. and Phillips. R. S.. Immunology 1978. 34: 821. 10 Brake, D. A., Long, C. A. and Weidanz, W. P., J. Immunol. 1988. 140: 1989. 11 Meding, S., Cheng, S. C., Simon-Haarhaus, B. and Langhorne, J., Infect. Immun. 1990. 58: 3671. 12 Langhorne, J.. Simon-Harhaus, B. and Meding, S.J., Immunol. Lett. 1990. 25: 101. 13 Bosma, G. C., Custer, R. I? and Bosma, M. J., Nature 1983.301: 527.
Eur. J. Immunol. 1991. 21: 1433-1438 14 Langhorne, J., Gillard, S., Simon. B., Slade, S. and Eichmann. K., Int. Immunol. 1989. I : 416. 15 Langhorne, J., Meding, S. J., Eichmann, K. and Gillard, S.. Immunol. Rev. 1989. 112: 71. 16 Julius, M. H., Simpson, E. and Herzenberg, L. A , , Eur. J. Immunol. 1973. 3: 645. 17 Cobbold, S. I?, Jayasuriya, A., Nash, A., Prospero,T. D. and Waldmann, H., Nature 1984. 312: 548. 18 Bruce, J., Symington. F. W., McKearn, T. J. and Sprent, J.. J. Imrnunol. 1981. 127: 2496. 19 Ledbetter, J. A. and Herzenberg, L. A., Immunol. Rev. 1979. 47: 63. 20 Langhorne. J. and Simon, B., Parasit. Immunol. 1989. 11: 545. 21 Kincade, I? W., Lee, G., Watanabe, T., Sun, L. and Scheid, M.P., J. Immunol. 1981. 127: 2262. 22 Coffman, R. L., Immunol. Rev. 1982. 69: 5. 23 Unkeless, J. C., J. Exp. Med. 1979. 150: 580. 24 Langhorne, J. and Titus, J., Eur. J. Immunol. 1988. 18: 1. 25 Langhorne, J., Evans, C. B., Asofsky, R. and Taylor, D. W., Cell. Immunol. 1984. 87: 452. 26 Brinkmann,V., Kaufmann, S. H. E. and Simon, M. M., Infect. Immun. 1985. 47: 737. 27 Snapper, C. M. and Paul, W. E., Science 1987. 236: 944. 28 Horowitz, J. B., Kay, J., Conrad, P. J., Katz, M. E. and Janeway, C. A,, Proc. Natl. Acad. Sci. USA 1986. 83: 1886. 29 Fiorentino, D. F., Bond, M. W. and Mosmann,T. R., J. Exp. Med. 1989. 170: 2081. 30 OGarra, A., Stapleton. G., Dhar,V., Pearce, M., Schumacher, J., Rugo, H., Barbis, B., Stall,A., Cupp, J., Moore, K.,Vieira, €?, Mosmann,T. R.,Whitmore, A., Arnold, L., Haughton, G. and Howard, M., Infect. Immun. 1990. 2: 821. 31 Kim, K. J., Rollwagen, F., Asofsky. R. and Lefkovits, I.. Eur. J. Immunol. 1984. 14: 476. 32 Grun, J. L., Long, C. A. and Weidanz,W. I?, Infect. Immun. 1985. 48: 853.
