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Lipopolysaccharides can convert heterologous red cells into thymus-independent antigens
CELLULAR
IMMUNOLOGY
4, 416424 (1972)
Lipopolysaccharides
Can
into G~RAN
Convert
Heterologous
Thymus-Independent
Antigens
MILLER,
JAN ANDERSON,
AND OLOF
Red Cells SJ~BERG
Division of Imnzunobiolog)!, Karollnska Institutet, Wallenberglaboratory, Lilla Freskati, 104 0.5 Stockholm 50, Sweden; and Base1 Institute for Immulzology, 487, Grenzacherstrasse, CH-40.58 Basel, Switzerland Xeceimd
December
2, 1971
Heterologous red cells coated with lipopolysaccharides (LPS) from E. coli 0127 bacteria induced a normal immune response to the red cell antigen in thymectomized, lethally irradiated, and bone marrow repopulated mice (T X B mice) ; whereas uncoated red cells only stimulated a weak response. Lipopolysaccharides had to be present on the red cells to convert them to thymus-independent antigens. T X B mice pretreated with large doses of LPS, in order to induce immunological tolerance, responded to LPS-coated red cells better than nonthymectomized mice to red cells alone. Passive transfer of antibody to LPS suppressed the response to red cells in T X B mice injected with LPS-coated red cells. Since LPS has been found to be a mitogen for bone marrow-derived (B) cells, it is suggested that this property is responsible for the ability of LPS to substitute for thymus-derived (T) cells. Consequently, the signal from T to B cells may be nonspecific. INTRODUCTION
Certain antigens, such as heterologous proteins and red cell antigens require the participation of both thymus-derived (T) and bone-marrow-derived (B) cells for induction of antibody synthesis in the B cells (1). Other antigens, such as pneumococcal polysaccharides, lipopolysaccharides (LPS) f rom E. coli bacteria and polyvinylpyrolidone (PVP) are thymus independent and give rise to antibody production in the absence of T cells (Z-4). In the case of LPS it has been clearly demonstrated that immune mice do not possess antigen-binding T cells (5) ; whereas antigen-binding T cells are consistently present against thymus-dependent antigens in normal or immune mice (6). The immune response against LPS is differentiated from that against
T-dependent
antigens
by the persistent
IgM
antibody
syn-
thesis and the more or less complete absence of an IgG antibody synthesis, as well as by the poor development of immunological memory (7). The T independence of the immune response to LPS may be ascribed to one of three possibilities (a) A B-B cell cooperation may occur with this particular antigen. (b) B cells may be nonspecifically stimulated by LPS (a mitogenic effect) or, finally, (c) LPS may have the ability of activating specific antigen-sensitive by itself, by, e.g., the formation of multiple bonds clue to the structure
being made up of repeating antigenic determinants 416 Copyright All rights
0 1972 by Academic Press. of reproduction in any form
Inc. reserved.
B cells of LPS,
[for discussion see (8) 1. In an
THYMUS
INDEPENDENCY
OF RED
CELLS
417
attempt to distinguish between these alternatives, it was studied whether LPS from E. coli bacteria coupled to sheep or horse red cells would convert the T-dependent red cell antigens into T-independent antigens. This would be expected to occur according to the second but not the third alternative above. The first possibility could not be excluded irrespective of the results of this type of experiment. MATERIALS AND METHODS
Mice. Mice of the inbred strain CBA, A, C57BL, C57L, and 5M as well as F, hybrids between these strains were used. In addition, the noninbred strain NMRI was employed. The mice were between 2 and 5 months old and of both sexes. Antigens and immunization. Sheep red blood cells (SRBC) or horse red blood cells (HRBC) were stored in Alsever’s solution. For immunization, 4 X 10’ sheep cells were injected intravenously in a volume of 0.2 ml. Bacterial antigens from E. coli 0127 (LPS) were also employed. Endotosin from these bacteria were extracted according to the methods of Westphal, Liiderlitz. and Bister (9) and detoxified as described before (10). LPS-coated red cells were used for immunization and they were prepared as described below. Cell suspensions. Cell suspensions were prepared by pressing spleens through a 60-mesh steel screen into balanced salt solution (BSS). Cell clumps were removed by filtrations through gauze. The number of trypan blue unstained cells were counted in a haemocytometer and the desired cell concentration was made up by dilution in BSS. Bone marrow cells were obtained from the femurs and tibia of mice. The bones were cut at both ends and the cells were removed by flushing the bone marrow cavity with BSS. The cells were suspended by repeated suctions in a pipette, thereafter filtered through gauze and subsequently treated as described for spleen cells. Thymus cell suspensions were obtained by pressing thymus organs through a 60-mesh stainless-steel screen as described for spleen cells. Detection of antibody-producing cells. These cells were detected by the agar plaque technique ( 11) with the modifications described by Mishell and Dutton (12), In short, the agar plaque technique was performed in 0.7% agar added on to cover glasses, which were incubated for 1 hr at 37” in a humid atmosphere. Thereafter, guinea pig serum diluted 1 :lO was added as a source of complement and incubation continued for another hour. Cellular antibody production to endotoxin was studied as described before (7). In short, polysaccharides were used to coat RBC; and the antigen-coated red cells were employed in the agar plaque technique. Coating was achieved by incubating 1 mg of endotoxin in 1 ml with 0.1 ml of packed RBC for 30 min at 37”. Thereafter, the RBC were washed three times and made up to a concentration of 25% in BSS. Thymes dependence. The dependence on thymus cells of the immune response against RBC, was studied in X-irradiated (700 R) syngeneic mice. The animals were untreated or had been previously thymectomized. Groups of five mice received 20 X lo6 bone marrow cells from untreated donors. After 20 to 30 days they were immunized with RBC or LPS-coated red cells intravenously. Five days later the spleens were removed and the number of antibody-producing cells to the red cell antigen was determined.
418
MCLLER,
ANDERSSON,
AND
SJijBERG
Tolerance induction. Tolerance was induced by 4 to 5 injections of 2 mg of alkali detoxified LPS with 2 to 3 days interval. Three days after the last injection the animals were immunized with LPS-coated red cells. X-Irradiation. Mice were X-irradiated at a rate of 62 R/min. X-Rays were generated in a Siemens X-ray machine at 200 kV and 15 mA and were filtered through 0.5 mm Cu. RESULTS
Thymus-Independence of LPS-Coated Red Cells Sheep or horse red blood cells were coated with LPS and thereafter washed. The cells were inoculated into T X B mice and, as a control, into animals which had not been thymectomized, but lethally irradiated and bone marrow repopulated. Uncoated red blood cells were injected alone in both groups to establish the normal response to the red cells. Furthermore, uncoated red blood cells were inoculated intravenously followed by an optimally immunizing LPS dose (0.01) intraperitoneally, to establish the nonspecific stimulatory effect of LPS on the immune response to heterologous red cells. The immune response was always tested against the heterologous red cells. The results are shown in Table 1 and an example in TA4BLE THYMUS
Strain
INDEPENDENCE
Treatment of animals
I
OF HETEROLOGOUS
RED
CELLS
COATED
TXBb
N x Be
A X 5M
TXB NXB
A X C57L
TXB NXB
SRBC SRBC-LPS SRBC+LPS SRBC SRBC-LPS SRBC+LPS
LPS (1
19s PFC response log, 0 mean + SE (antilog) against the red cells per:
Antigen
Spleen A X 5M
WITH
106/spleen cells
3.56=tO.O9 4.44f0.03 3.34hO.39 4.85ZtO.04 4.99Zto.07 4.91f0.08
(3.669) (27.394) (2.188) (71.179) (98.213) (81.775)
1.17I!Zo.o5 2.19xtO.05 1.091tO.38 2.21~kO.08 2.48+0.05 2.34kO.07
(15) (153) (12) (162) (299) (220)
HRBC H RBC - LPS HRBC HRBC - LPS
2.20xt0.22 3.48*0.09 3.17f0.06 3.84f0.13
(167) (3.007) (1.485) (6.853)
0.395~0.20 1.69fO. 11 1.23xtO.09 1.86~kO. 15
(2) (49) (17) (72)
HRBC HRBC-LPS HRBC HRBC-LPS
2.37f0.23 3.26f0.09 3.46f0.09 3.67~tO.09
(233) (1.811) (2.900) (4.691)
0.425~0.13 1.375~0.13 1.34&O. 12 1.611t0.21
(3) (23) (22) (41)
c *
a Thymectomized or nonthymectomized mice were lethally irradiated and repopulated with syngeneic bone marrow cells. Thereafter they were injected intravenously with E. coli 0127 lipopolysaccharide-coated heterologous red cells. The plaque-forming response to the red cells was determined 5 days later. b T X B = thymectomized, lethally irradiated and bone marrow repopulated mice. c SRBC-LPS = E. coli 0127 lipopolysaccharide-coated sheep red cells injected intravenously. d SRBCfLPS = E. coli lipopolysaccharide (0.01 mg) injected intraperitoneally and sheep red cells injected intravenously. e N X B = nonthymectomized, lethally irradiated and bone marrow repopulated mice.
THYMUS
INDEPENDENCY
OF RED
CELLS
419
Fig, 1. As shown, the immune response to the heterologous red blood cells was highly thymus dependent and only a weak immune response was induced in T X B mice, as compared to the response obtained in nonthymectomized mice. HOWever, if the red blood cells were coated with LPS there was a marked enhancement of the number of plaque-forming cells (PFC) against the red cells in T X B mice. Generally the PFC response increased 10 times and reached levels comparable to those found in nonthymectomized animals (Table 1). This was not due to a generally stimulating effect of LPS on antibody synthesis, because LPS given by a separate route did not cause stimulation of the PFC response to red cells in either T X B or nonthymectomized mice. Thus, heterologous red blood cells can be converted into thymus-independent antigens by coating them with LPS. It was essential that the LPS was coated onto the red cell, because no effect was seen when the two antigens were given by separate routes. Effect of Pretreating Animals with Large noses of LPS AAs outlined in the introduction, there exist several possibilities to explain the finding that LPS-coated red cells become thymus independent. Since there are no T cells against LPS in immune animals, it cannot be argued that LPS acts as a carrier reacting with T cells in this way causing a T-B cell cooperation. However, it seemed plausible that an immune response to LPS may have helped present the LPS-coated red cells to the B cell in an optimally immunogenic form. Several experiments have shown that antibodies to a certain antigen can amplify the immune response and even transform haptens into immunogens (13-15). To test whether antibodies to the LPS were responsible for the T independence of the LPS-coated red cells, attempts were made to induce immunological tolerance to LPS by injecting animals with S-10 mg of LPS with 2-3 days’ interval, a procedure known to induce specific immunological paralysis (16). In the present experiments tolerance induction was induced in lethally irradiated and bone marrow repopulated mice. Tolerance by treatment was not complete, but was revealed as a depressed number of PFC against LPS. In the experiments T X B as well as nonthymectomized, but otherwise similarly treated, mice were divided into two groups, one being untreated, whereas the other was pretreated with large doses of LPS. Both groups were further divided into
Thymectombed
non-thymectom~zed
1. PFC response against HRBC in thymectomized or nonthymectomized, lethally irradiated and bone marrow repopulated mice injected with the indicated cells : (RBC-etox) FIG.
red cells coated with lipopolysaccharide of E. coli 0127 origin; lipopolysaccharide were injected separately by different routes.
(RBC -I- etox) red cells and
420
MijLLER,
ANDERSSON,
TABI,E
AND
SJijBERG
2
THYMUS INDEPENDENCE OF LPS-COATED HETEKOLOGOUS RED CELLS IN T X B MICE PRETREATED WITH 8 TO 10 mg LPSa
Strain A x C57BL
Treatment of animals TXB
Antigen injected
-
HRBC HRBC-LPS HRBC HRBC-LPS
1.82f0.12 2.85ztO.24 2.52&O. 17 3.34ztO.06
(66) (711) (331) (2.182)
0.081tO. 17 1.04f0.23 0.78&O. 18 1.57ztO.08
(1) (11) (6) (37)
HRBC HRBC-LPS HRBC-LPS HRBC HRBC-LPS HRBC-LPS
1.84f0.12 3.22zt0.11 3.57ztO.22 2.75ztO. 11 3.06zkO.09 3.18f0.08
(68) (1.645) (3.752) (562) (1.145) (1.520)
O.OSf0.25 1.49f0.19 1.39xkO.24 1.06ztO. 16 1.34&O. 10 1.27zbO.07
(1) (31) (24) (11) (22) (19)
HRBC HKBC-LPS HRBC HRBC-LPS HRBC HRBC-LPS HRBC HRBC-LPS
1.72f0.36 2.55ztO.40 2.69xk0.26 3.11k0.39 2.831kO.22 3.71zk0.17 2.72zbO.28 3.34zkO.20
(52) (359) (491) (1.294) (674) (5.134) (520) (2.200)
o.oozto.35 0.83ztO.51 0.70f0.31 1.32zkO.37 0.821kO.24 1.64zk0.15 l.lOztzO.23 1.47=!=0.19
(0.9) (5) (5) (21) (8) (43) (13) (29)
+ + C57BL
TXB
NXB
+ +
A x C57BL
TXB
NXB
19s PFC log,, mean + SE (antilog) against the red cells per:
Pretreatment with LPS
+ + + +
Spleen
10B/cells
a The mice were given 2 mg of detoxified LPS intravenousI> 7 at 4 to 5 times with 3-4 days intervals. They \Nere immunized 3-4 days after the last injection with I*PS-coated horse red cells.
two subgroups, one given red cells alone, the other receiving LPS coated red cells. The results were analogous in all experiments as shown in Table 2 and Fig. 2. In T X B mice, red cells alone caused a weak immune response, which was enhanced, as shown before, by coating the red cells with LPS. If the T X B mice were pretreated with LPS to induce tolerance, the immune response to red blood cells alone was increased compared to the response found in nonpretreated animals. However, in LPS-pretreated animals, LPS-coated red cells caused an immune response to 40,
RBC RBC-etox RBC R%C-etox non-pretreated pretreated with etax
FIG. 2. PFC response against HRBC in T X B mice which were either previously untreated or injected with 10 mg of detoxified lipopolysaccharide prior to injection of the indicated red cells.
THYMUS
INDEPENDENCY
OF
RED
CEL1.S
421
the red cells that was markedly enhanced and comparable to that found in nonthymectomized mice given LPS-coated red cells. Although this effect, was very clearly seen in T X B mice, it was also observed to some extent in untreated animals. Thus, pretreatment of mice with LPS to induce partial immunological unresponsiveness, resulted in a more pronounced T-cell independence of LPS-coated red cells than in nonpretreated animals. Efect of Antibodies to Endotoxin The previous findings do not support the possibility that an immune response to LPS is responsible for the T-cell independence of red cells after LPS coatiing. However, tolerance to LPS results in an increased number of LPS binding B cells, even though the PFC response is suppressed (5). Therefore, experiments were performed with passive transfer of antibody to LPS to test whether this would amplify the response to the red cells specifically after injection of LPS-coated red cells. These experiments were performed in T X B mice, as well as in nonthymectomized, but otherwise similarly treated animals, which were given untreated red cells or LPS-coated red cells. Some animals in addition received specific antibody to the LPS or, as a control, antibody to other bacterial lipopolysaccharide antigens (from a different E. coli strain). The antibodies were taken from mice immunized once, 5 to 15 days prior to the experiments. As shown in Table 3, passive transfer of specific antibodies caused a suppression of the number of PFC to red cells compared to control groups treated with antibodies to other LPS strains. DISCUSSION
The present findings show that the immune response to red cell antigens, which is highly thymus dependent, can be made thymus independent by coating the red cells with LPS, which by itself is a thymus-independent antigen. The degree of thymus independence of the immune response to the red cells caused by LPS coating increased when the animals were pretreated with large doses of LPS, aiming at inducing specific tolerance and decreased when antibodies to LPS were administered passively. Preliminary findings by Andersson and Blomgren (3) also demonstrated that LPS-coated red cells became T independent. There was little effect by coating PVP, another T-independent antigen onto red cells, unless the recipients were given lymphocytes sensitized against PVP. There seems to be two main mechanisms by which the immune response to red cells become T independent after coating the red cells with LPS : (a) LPS-coated red cells may cause a cellular collaboration, known to be essential for the induction of an immune response to heterologous red cells ( 1). (b) LPS has a direct mitogenie effect on B cells that bind specifically the red cell antigens. a. The immune response to red cells requires collaboration between T and B cells. Therefore, there is a very weak response in T X B mice. It seems plausible that LPS coating may cause some type of collaboration between the antigen-sensitive B cells and other cells present in T X B mice. Such collaboration could occur between U cells and residual T cells or between two B cells. It is unlikely that a T and B cell collaboration occurs. because there are no detectable LPS l)inclillg T cells in immunized mice (5). However. it is possible that a B and B cell collabora-
of animals injected
HRBC HRBC-LPS HRBC-LPS HRBC-LPS
T X B
CBA Anti-0127 Anti-0127
Anti-B Anti-0127
Anti-0127
Anti-0127
Anti-0127 -
-
Anti-B Anti-0127
Anti-B h AIlti-0127 c
Specificity
Antibody
l/100 1/lOOO
1 /so0 l/500
l/500
l/500
l/500
l/500 l/500
l/500 l/500
Dilution
.3.15 3.86 3.18 3.24
3.20 3.92 3.79 3.34
4.19 4.37 4.08
3.77 4.42 4.02 4.35 4.51 4.46
3.63 4.49 3.88 4.75 4.67 4.62
Spleen
* z!z f f
xt 2~ + z!r
0.03 0.16 0.30 0.07
0.21 0.11 0.19 0.20
f 0.22 zt 0.19 f 0.12
0.19 0.14 0.12 0.09 0.13 0.11
f 0.12 + 0.11 zk 0.30 f 0.11 31 0.08 f 0.04 f f 3~ zt + zt
RED CELLS”
(1.412) (7.264) (1.498) (2.208)
(1.582) (8.245) (6.191) (2.198)
(15.631) (23.642) (12.062)
(5.908) (27.603) (10.468) (22.403) (32.239) (28.742)
(4.300) (31.223) (7.568) (56.331) (47.232) (41.639)
1.43 2.07 1.69 1.87
1.40 1.93 1.86 1.43
1.97 2.15 1.95
1.55 2.25 1.68 2.12 2.15 2.23
1.37 2.29 1.72 2.64 2.41 2.28
0.21 0.17 0.15 0.15 0.19 0.10
0.13 0.13 0.36 0.28 0.13 0.04
f f f +
zt zt f f
0.16 0.12 0.17 0.10
0.09 0.11 0.25 0.13
3z 0.25 It 0.13 + 0.10
zt i + zt Yt zt
xt zt i f zt I!=
(31) (118) (49) (74)
(25) (85) (73) (27)
(93) (141) (88)
(36) (176) (48) (131) (141) (170)
(24) (195) (53) (436) (256) (190)
lo6 spleen cells
19s PFC response to the red cells [log10 mean + SE (antilog)]
OF LPS-COATED
” The NMRI mice were thymectomized and thereafter irradiated with one leg shielded by lead. CBA mice were repopulated with bone marrow inoculation. The mice were immunized with red cells iv and given antibody ip 1 hr later. In all experiments, except the last, the immune serum was taken 5-15 days after a primary immunization. In the last experiment, hyperimmune serum \vas used. h i\nti-B = antiserum against E. coli B. c Anti-0127 = antiserum against E. coli 0127.
HRBC HRBC-LPS H RBC-LPS HRBC-LPS
‘I’ x B
NMKI
HRBC HRBC-LPS HRBC-LPS
HRBC HRBC-LPS HRBC-LPS HRBC HRBC-LPS HRBC-LPS
HRBC HRBC-LPS HRBC-LPS HRBC H RBC-LPS HRBC-LPS
Antigen
‘I‘ X B
NXB
‘I‘ X B
N x B
TXB
Treatment
NMRI
NMRI
NMRI
Strain
3
EFFECT OF ANTIBODIES TO LPS FROM E. coli 0127 ON THE THYMUS-INDEPENDENCE
TABLE
THYMUS
INDEPENDENCY
OF RED
CELLS
423
tion may be responsible. Such an interpretation is compatible with the tolerance experiments, because it has been shown that tolerance induction to LPS results in an increased number of LPS-binding cells (5)) and the antibody transfer experiments. because antibodies would prevent LPS from interacting with the B cells. However, there is no evidence for a B-B cell interaction in the immune response. Therefore, this explanation remains as a theoretical, but so far unsupported, possibility. b. LPS is a conventional immunogen and is competent to induce specific antibody synthesis, as well as specific tolerance. However, it has been clearly demonstrated that LPS can also function as a nonspecific mitogen for B cells, but not for T cells (17, 18). Thus, LPS has the ability to stimulate DNA synthesis in B cells and it can also activate antibody producing cells to various noncross reacting antigens, as if specific antigen had been added (19). Finally, LPS can selectively stimulate IgM synthesis in vitro (20). The B cell mitogenic property of LPS may explain the present results. Thus, when LPS-coated red cells have reacted with B precursor cells having immunoglobulin receptors for the red cells, LPS will have the possibility to act as a mitogen for these cells and thereby activate them to division and differentiation, even in the absence of a T-B cell cooperation. The results of tolerance induction to LPS would also be compatible with this interpretation because the large amount of LPS injected will persist undegraded for a long time in the animals and can bind nonspecifically to many B cells. When these animals are injected with LPS-coated red cells, those B cells that recognize the red cells by immunoglobulin receptors are confronted with a specific antigenic st,imulus, by itself insufficient to activate the cells and, in addition, a large number of LPS molecules, contributed both by those presenb on the red cells and those previously injected. This would cause a stronger nonspecific stimulus to division than if the animals had not been pretreated. Passive transfer of antibody to LPS would decrease its ability to interact with the B lymphocytes and therefore this treatment would clecrease the capacity of LPS to stimulate the response to LPS-coated red cells. The second interpretation of the results cannot be formally proved, but is in agreement with the in vitro findings mentioned above, showing that LPS can activate B cells nonspecifically. It has also been demonstrated that, LPS can substitute for T cells in the induction of an immune response in vitro to red cell antigens (19). It follows that LPS can supplement for the T cell function, which helps activating B cells in the immune response. Therefore, the T-B cell cooperation may be nonspecific in nature, because the T cell signal to B cells may be substituted by a nonspecific B cell mitogen. Recent findings is in agreement with this postulate and demonstrate that at least one T cell signal to the B cells can be a nonspecific humoral factor, which by itself does not act as a mitogen, but in cooperation with an inducing agent (antigen or mitogen) can cause proliferation of B cells (21 1. The T cell independence of the immune response to LPS itself may be caused by the dual function of LPS, being both an antigen and a nonspecific mitogen. However, nonmitogenic antigens, such as PVP and pneunlococcal polysac&ari&s, are also T cell independent. Therefore, the B-B cell cooperation and the ability of these antigens to activate B cells directly by, e.g.. multiple bond formation still exists as plausible interpretations for their T cell independence.
424
MGLLER,
ANDERSSON,
AND
SJijBERG
ACKNOWLEDGMENTS The part of this work carried out in Sweden was supported by grants from the Swedish Medical Research Council, the Swedish Cancer Society, the Wallenberg Foundation and the Harald Jeansson’s Foundation. The technical assistance of Mrs. Kerstin Andersson is gratefully acknowledged. REFERENCES 1. Transplant.
Rev. 1, 1, 1969.
2. Miiller, G., and Michael, G., Cell. Ia2+nnxol. 2, 309, 1971. 3. Andersson, B., and Blomgren, H., Cell. I~mmtlzol. 2, 411, 1971. 4. Howard, J. G., Christie, G. H., Courtenay, B. M., Leuchars, E., and Davies, .4. J. S., Cell. Imnzunol. 2, 614, 1971. 5. Sjijberg, O., J. Exp. Med. 133, 1015, 1971. 6. Greaves, M. F., and Moller, E., Cell. Immunol. 1, 372, 1970. 7. Mijller, G., Nature (London) 207, 1166, 1963. 8. Moller, G., Cell. Imnmnol. 1, 573, 1970. 9. Westphal, O., Liiderlitz, O., and Bister, F., Z. Natwfors~h. 7, 148, 1952. 10. Britton, S., Immulzologv 16, 513, 1969. 11. Jerne, N. K., and Nordjn, A. A. S&ace 140, 405, 1963. 12. Mishell, R., and Dutton, R., J. Exp. Med. 123, 423, 1967. 13. Henry, C., and Jerne, N. K., J. Exp. Med. 126, 133, 1968. 14. Feldman, M., and Diener, E., J. Exp. Med. 131, 247, 1970. 15. McBride, R. A., and Schierman, L., J. Exp. Med. 131, 377, 1970. 16. Britton, S., Immulzology 16, 527, 1969. 17. Peavy, D. L., Adler, W. H., and Smith, R. T., J. Immunol. 105, 1453, 1970. 18. Andersson, J., Moller, G., and Sjgberg, O., Cell. Immzlnol. 3, 000, 1972. 19. Sjoberg, O., i2nderssoq J., and Moller, G., Europ. J. Imnwnology, In press. 20. .4ndersson, J,, Moller, G., and Sjoberg, O., Europ. J. Imnrzmology, In press. 21. Andersson, J., Miiller, G., and SjBberg, O., Eur. J. Immwology. In press.
IMMUNOLOGY
4, 416424 (1972)
Lipopolysaccharides
Can
into G~RAN
Convert
Heterologous
Thymus-Independent
Antigens
MILLER,
JAN ANDERSON,
AND OLOF
Red Cells SJ~BERG
Division of Imnzunobiolog)!, Karollnska Institutet, Wallenberglaboratory, Lilla Freskati, 104 0.5 Stockholm 50, Sweden; and Base1 Institute for Immulzology, 487, Grenzacherstrasse, CH-40.58 Basel, Switzerland Xeceimd
December
2, 1971
Heterologous red cells coated with lipopolysaccharides (LPS) from E. coli 0127 bacteria induced a normal immune response to the red cell antigen in thymectomized, lethally irradiated, and bone marrow repopulated mice (T X B mice) ; whereas uncoated red cells only stimulated a weak response. Lipopolysaccharides had to be present on the red cells to convert them to thymus-independent antigens. T X B mice pretreated with large doses of LPS, in order to induce immunological tolerance, responded to LPS-coated red cells better than nonthymectomized mice to red cells alone. Passive transfer of antibody to LPS suppressed the response to red cells in T X B mice injected with LPS-coated red cells. Since LPS has been found to be a mitogen for bone marrow-derived (B) cells, it is suggested that this property is responsible for the ability of LPS to substitute for thymus-derived (T) cells. Consequently, the signal from T to B cells may be nonspecific. INTRODUCTION
Certain antigens, such as heterologous proteins and red cell antigens require the participation of both thymus-derived (T) and bone-marrow-derived (B) cells for induction of antibody synthesis in the B cells (1). Other antigens, such as pneumococcal polysaccharides, lipopolysaccharides (LPS) f rom E. coli bacteria and polyvinylpyrolidone (PVP) are thymus independent and give rise to antibody production in the absence of T cells (Z-4). In the case of LPS it has been clearly demonstrated that immune mice do not possess antigen-binding T cells (5) ; whereas antigen-binding T cells are consistently present against thymus-dependent antigens in normal or immune mice (6). The immune response against LPS is differentiated from that against
T-dependent
antigens
by the persistent
IgM
antibody
syn-
thesis and the more or less complete absence of an IgG antibody synthesis, as well as by the poor development of immunological memory (7). The T independence of the immune response to LPS may be ascribed to one of three possibilities (a) A B-B cell cooperation may occur with this particular antigen. (b) B cells may be nonspecifically stimulated by LPS (a mitogenic effect) or, finally, (c) LPS may have the ability of activating specific antigen-sensitive by itself, by, e.g., the formation of multiple bonds clue to the structure
being made up of repeating antigenic determinants 416 Copyright All rights
0 1972 by Academic Press. of reproduction in any form
Inc. reserved.
B cells of LPS,
[for discussion see (8) 1. In an
THYMUS
INDEPENDENCY
OF RED
CELLS
417
attempt to distinguish between these alternatives, it was studied whether LPS from E. coli bacteria coupled to sheep or horse red cells would convert the T-dependent red cell antigens into T-independent antigens. This would be expected to occur according to the second but not the third alternative above. The first possibility could not be excluded irrespective of the results of this type of experiment. MATERIALS AND METHODS
Mice. Mice of the inbred strain CBA, A, C57BL, C57L, and 5M as well as F, hybrids between these strains were used. In addition, the noninbred strain NMRI was employed. The mice were between 2 and 5 months old and of both sexes. Antigens and immunization. Sheep red blood cells (SRBC) or horse red blood cells (HRBC) were stored in Alsever’s solution. For immunization, 4 X 10’ sheep cells were injected intravenously in a volume of 0.2 ml. Bacterial antigens from E. coli 0127 (LPS) were also employed. Endotosin from these bacteria were extracted according to the methods of Westphal, Liiderlitz. and Bister (9) and detoxified as described before (10). LPS-coated red cells were used for immunization and they were prepared as described below. Cell suspensions. Cell suspensions were prepared by pressing spleens through a 60-mesh steel screen into balanced salt solution (BSS). Cell clumps were removed by filtrations through gauze. The number of trypan blue unstained cells were counted in a haemocytometer and the desired cell concentration was made up by dilution in BSS. Bone marrow cells were obtained from the femurs and tibia of mice. The bones were cut at both ends and the cells were removed by flushing the bone marrow cavity with BSS. The cells were suspended by repeated suctions in a pipette, thereafter filtered through gauze and subsequently treated as described for spleen cells. Thymus cell suspensions were obtained by pressing thymus organs through a 60-mesh stainless-steel screen as described for spleen cells. Detection of antibody-producing cells. These cells were detected by the agar plaque technique ( 11) with the modifications described by Mishell and Dutton (12), In short, the agar plaque technique was performed in 0.7% agar added on to cover glasses, which were incubated for 1 hr at 37” in a humid atmosphere. Thereafter, guinea pig serum diluted 1 :lO was added as a source of complement and incubation continued for another hour. Cellular antibody production to endotoxin was studied as described before (7). In short, polysaccharides were used to coat RBC; and the antigen-coated red cells were employed in the agar plaque technique. Coating was achieved by incubating 1 mg of endotoxin in 1 ml with 0.1 ml of packed RBC for 30 min at 37”. Thereafter, the RBC were washed three times and made up to a concentration of 25% in BSS. Thymes dependence. The dependence on thymus cells of the immune response against RBC, was studied in X-irradiated (700 R) syngeneic mice. The animals were untreated or had been previously thymectomized. Groups of five mice received 20 X lo6 bone marrow cells from untreated donors. After 20 to 30 days they were immunized with RBC or LPS-coated red cells intravenously. Five days later the spleens were removed and the number of antibody-producing cells to the red cell antigen was determined.
418
MCLLER,
ANDERSSON,
AND
SJijBERG
Tolerance induction. Tolerance was induced by 4 to 5 injections of 2 mg of alkali detoxified LPS with 2 to 3 days interval. Three days after the last injection the animals were immunized with LPS-coated red cells. X-Irradiation. Mice were X-irradiated at a rate of 62 R/min. X-Rays were generated in a Siemens X-ray machine at 200 kV and 15 mA and were filtered through 0.5 mm Cu. RESULTS
Thymus-Independence of LPS-Coated Red Cells Sheep or horse red blood cells were coated with LPS and thereafter washed. The cells were inoculated into T X B mice and, as a control, into animals which had not been thymectomized, but lethally irradiated and bone marrow repopulated. Uncoated red blood cells were injected alone in both groups to establish the normal response to the red cells. Furthermore, uncoated red blood cells were inoculated intravenously followed by an optimally immunizing LPS dose (0.01) intraperitoneally, to establish the nonspecific stimulatory effect of LPS on the immune response to heterologous red cells. The immune response was always tested against the heterologous red cells. The results are shown in Table 1 and an example in TA4BLE THYMUS
Strain
INDEPENDENCE
Treatment of animals
I
OF HETEROLOGOUS
RED
CELLS
COATED
TXBb
N x Be
A X 5M
TXB NXB
A X C57L
TXB NXB
SRBC SRBC-LPS SRBC+LPS SRBC SRBC-LPS SRBC+LPS
LPS (1
19s PFC response log, 0 mean + SE (antilog) against the red cells per:
Antigen
Spleen A X 5M
WITH
106/spleen cells
3.56=tO.O9 4.44f0.03 3.34hO.39 4.85ZtO.04 4.99Zto.07 4.91f0.08
(3.669) (27.394) (2.188) (71.179) (98.213) (81.775)
1.17I!Zo.o5 2.19xtO.05 1.091tO.38 2.21~kO.08 2.48+0.05 2.34kO.07
(15) (153) (12) (162) (299) (220)
HRBC H RBC - LPS HRBC HRBC - LPS
2.20xt0.22 3.48*0.09 3.17f0.06 3.84f0.13
(167) (3.007) (1.485) (6.853)
0.395~0.20 1.69fO. 11 1.23xtO.09 1.86~kO. 15
(2) (49) (17) (72)
HRBC HRBC-LPS HRBC HRBC-LPS
2.37f0.23 3.26f0.09 3.46f0.09 3.67~tO.09
(233) (1.811) (2.900) (4.691)
0.425~0.13 1.375~0.13 1.34&O. 12 1.611t0.21
(3) (23) (22) (41)
c *
a Thymectomized or nonthymectomized mice were lethally irradiated and repopulated with syngeneic bone marrow cells. Thereafter they were injected intravenously with E. coli 0127 lipopolysaccharide-coated heterologous red cells. The plaque-forming response to the red cells was determined 5 days later. b T X B = thymectomized, lethally irradiated and bone marrow repopulated mice. c SRBC-LPS = E. coli 0127 lipopolysaccharide-coated sheep red cells injected intravenously. d SRBCfLPS = E. coli lipopolysaccharide (0.01 mg) injected intraperitoneally and sheep red cells injected intravenously. e N X B = nonthymectomized, lethally irradiated and bone marrow repopulated mice.
THYMUS
INDEPENDENCY
OF RED
CELLS
419
Fig, 1. As shown, the immune response to the heterologous red blood cells was highly thymus dependent and only a weak immune response was induced in T X B mice, as compared to the response obtained in nonthymectomized mice. HOWever, if the red blood cells were coated with LPS there was a marked enhancement of the number of plaque-forming cells (PFC) against the red cells in T X B mice. Generally the PFC response increased 10 times and reached levels comparable to those found in nonthymectomized animals (Table 1). This was not due to a generally stimulating effect of LPS on antibody synthesis, because LPS given by a separate route did not cause stimulation of the PFC response to red cells in either T X B or nonthymectomized mice. Thus, heterologous red blood cells can be converted into thymus-independent antigens by coating them with LPS. It was essential that the LPS was coated onto the red cell, because no effect was seen when the two antigens were given by separate routes. Effect of Pretreating Animals with Large noses of LPS AAs outlined in the introduction, there exist several possibilities to explain the finding that LPS-coated red cells become thymus independent. Since there are no T cells against LPS in immune animals, it cannot be argued that LPS acts as a carrier reacting with T cells in this way causing a T-B cell cooperation. However, it seemed plausible that an immune response to LPS may have helped present the LPS-coated red cells to the B cell in an optimally immunogenic form. Several experiments have shown that antibodies to a certain antigen can amplify the immune response and even transform haptens into immunogens (13-15). To test whether antibodies to the LPS were responsible for the T independence of the LPS-coated red cells, attempts were made to induce immunological tolerance to LPS by injecting animals with S-10 mg of LPS with 2-3 days’ interval, a procedure known to induce specific immunological paralysis (16). In the present experiments tolerance induction was induced in lethally irradiated and bone marrow repopulated mice. Tolerance by treatment was not complete, but was revealed as a depressed number of PFC against LPS. In the experiments T X B as well as nonthymectomized, but otherwise similarly treated, mice were divided into two groups, one being untreated, whereas the other was pretreated with large doses of LPS. Both groups were further divided into
Thymectombed
non-thymectom~zed
1. PFC response against HRBC in thymectomized or nonthymectomized, lethally irradiated and bone marrow repopulated mice injected with the indicated cells : (RBC-etox) FIG.
red cells coated with lipopolysaccharide of E. coli 0127 origin; lipopolysaccharide were injected separately by different routes.
(RBC -I- etox) red cells and
420
MijLLER,
ANDERSSON,
TABI,E
AND
SJijBERG
2
THYMUS INDEPENDENCE OF LPS-COATED HETEKOLOGOUS RED CELLS IN T X B MICE PRETREATED WITH 8 TO 10 mg LPSa
Strain A x C57BL
Treatment of animals TXB
Antigen injected
-
HRBC HRBC-LPS HRBC HRBC-LPS
1.82f0.12 2.85ztO.24 2.52&O. 17 3.34ztO.06
(66) (711) (331) (2.182)
0.081tO. 17 1.04f0.23 0.78&O. 18 1.57ztO.08
(1) (11) (6) (37)
HRBC HRBC-LPS HRBC-LPS HRBC HRBC-LPS HRBC-LPS
1.84f0.12 3.22zt0.11 3.57ztO.22 2.75ztO. 11 3.06zkO.09 3.18f0.08
(68) (1.645) (3.752) (562) (1.145) (1.520)
O.OSf0.25 1.49f0.19 1.39xkO.24 1.06ztO. 16 1.34&O. 10 1.27zbO.07
(1) (31) (24) (11) (22) (19)
HRBC HKBC-LPS HRBC HRBC-LPS HRBC HRBC-LPS HRBC HRBC-LPS
1.72f0.36 2.55ztO.40 2.69xk0.26 3.11k0.39 2.831kO.22 3.71zk0.17 2.72zbO.28 3.34zkO.20
(52) (359) (491) (1.294) (674) (5.134) (520) (2.200)
o.oozto.35 0.83ztO.51 0.70f0.31 1.32zkO.37 0.821kO.24 1.64zk0.15 l.lOztzO.23 1.47=!=0.19
(0.9) (5) (5) (21) (8) (43) (13) (29)
+ + C57BL
TXB
NXB
+ +
A x C57BL
TXB
NXB
19s PFC log,, mean + SE (antilog) against the red cells per:
Pretreatment with LPS
+ + + +
Spleen
10B/cells
a The mice were given 2 mg of detoxified LPS intravenousI> 7 at 4 to 5 times with 3-4 days intervals. They \Nere immunized 3-4 days after the last injection with I*PS-coated horse red cells.
two subgroups, one given red cells alone, the other receiving LPS coated red cells. The results were analogous in all experiments as shown in Table 2 and Fig. 2. In T X B mice, red cells alone caused a weak immune response, which was enhanced, as shown before, by coating the red cells with LPS. If the T X B mice were pretreated with LPS to induce tolerance, the immune response to red blood cells alone was increased compared to the response found in nonpretreated animals. However, in LPS-pretreated animals, LPS-coated red cells caused an immune response to 40,
RBC RBC-etox RBC R%C-etox non-pretreated pretreated with etax
FIG. 2. PFC response against HRBC in T X B mice which were either previously untreated or injected with 10 mg of detoxified lipopolysaccharide prior to injection of the indicated red cells.
THYMUS
INDEPENDENCY
OF
RED
CEL1.S
421
the red cells that was markedly enhanced and comparable to that found in nonthymectomized mice given LPS-coated red cells. Although this effect, was very clearly seen in T X B mice, it was also observed to some extent in untreated animals. Thus, pretreatment of mice with LPS to induce partial immunological unresponsiveness, resulted in a more pronounced T-cell independence of LPS-coated red cells than in nonpretreated animals. Efect of Antibodies to Endotoxin The previous findings do not support the possibility that an immune response to LPS is responsible for the T-cell independence of red cells after LPS coatiing. However, tolerance to LPS results in an increased number of LPS binding B cells, even though the PFC response is suppressed (5). Therefore, experiments were performed with passive transfer of antibody to LPS to test whether this would amplify the response to the red cells specifically after injection of LPS-coated red cells. These experiments were performed in T X B mice, as well as in nonthymectomized, but otherwise similarly treated animals, which were given untreated red cells or LPS-coated red cells. Some animals in addition received specific antibody to the LPS or, as a control, antibody to other bacterial lipopolysaccharide antigens (from a different E. coli strain). The antibodies were taken from mice immunized once, 5 to 15 days prior to the experiments. As shown in Table 3, passive transfer of specific antibodies caused a suppression of the number of PFC to red cells compared to control groups treated with antibodies to other LPS strains. DISCUSSION
The present findings show that the immune response to red cell antigens, which is highly thymus dependent, can be made thymus independent by coating the red cells with LPS, which by itself is a thymus-independent antigen. The degree of thymus independence of the immune response to the red cells caused by LPS coating increased when the animals were pretreated with large doses of LPS, aiming at inducing specific tolerance and decreased when antibodies to LPS were administered passively. Preliminary findings by Andersson and Blomgren (3) also demonstrated that LPS-coated red cells became T independent. There was little effect by coating PVP, another T-independent antigen onto red cells, unless the recipients were given lymphocytes sensitized against PVP. There seems to be two main mechanisms by which the immune response to red cells become T independent after coating the red cells with LPS : (a) LPS-coated red cells may cause a cellular collaboration, known to be essential for the induction of an immune response to heterologous red cells ( 1). (b) LPS has a direct mitogenie effect on B cells that bind specifically the red cell antigens. a. The immune response to red cells requires collaboration between T and B cells. Therefore, there is a very weak response in T X B mice. It seems plausible that LPS coating may cause some type of collaboration between the antigen-sensitive B cells and other cells present in T X B mice. Such collaboration could occur between U cells and residual T cells or between two B cells. It is unlikely that a T and B cell collaboration occurs. because there are no detectable LPS l)inclillg T cells in immunized mice (5). However. it is possible that a B and B cell collabora-
of animals injected
HRBC HRBC-LPS HRBC-LPS HRBC-LPS
T X B
CBA Anti-0127 Anti-0127
Anti-B Anti-0127
Anti-0127
Anti-0127
Anti-0127 -
-
Anti-B Anti-0127
Anti-B h AIlti-0127 c
Specificity
Antibody
l/100 1/lOOO
1 /so0 l/500
l/500
l/500
l/500
l/500 l/500
l/500 l/500
Dilution
.3.15 3.86 3.18 3.24
3.20 3.92 3.79 3.34
4.19 4.37 4.08
3.77 4.42 4.02 4.35 4.51 4.46
3.63 4.49 3.88 4.75 4.67 4.62
Spleen
* z!z f f
xt 2~ + z!r
0.03 0.16 0.30 0.07
0.21 0.11 0.19 0.20
f 0.22 zt 0.19 f 0.12
0.19 0.14 0.12 0.09 0.13 0.11
f 0.12 + 0.11 zk 0.30 f 0.11 31 0.08 f 0.04 f f 3~ zt + zt
RED CELLS”
(1.412) (7.264) (1.498) (2.208)
(1.582) (8.245) (6.191) (2.198)
(15.631) (23.642) (12.062)
(5.908) (27.603) (10.468) (22.403) (32.239) (28.742)
(4.300) (31.223) (7.568) (56.331) (47.232) (41.639)
1.43 2.07 1.69 1.87
1.40 1.93 1.86 1.43
1.97 2.15 1.95
1.55 2.25 1.68 2.12 2.15 2.23
1.37 2.29 1.72 2.64 2.41 2.28
0.21 0.17 0.15 0.15 0.19 0.10
0.13 0.13 0.36 0.28 0.13 0.04
f f f +
zt zt f f
0.16 0.12 0.17 0.10
0.09 0.11 0.25 0.13
3z 0.25 It 0.13 + 0.10
zt i + zt Yt zt
xt zt i f zt I!=
(31) (118) (49) (74)
(25) (85) (73) (27)
(93) (141) (88)
(36) (176) (48) (131) (141) (170)
(24) (195) (53) (436) (256) (190)
lo6 spleen cells
19s PFC response to the red cells [log10 mean + SE (antilog)]
OF LPS-COATED
” The NMRI mice were thymectomized and thereafter irradiated with one leg shielded by lead. CBA mice were repopulated with bone marrow inoculation. The mice were immunized with red cells iv and given antibody ip 1 hr later. In all experiments, except the last, the immune serum was taken 5-15 days after a primary immunization. In the last experiment, hyperimmune serum \vas used. h i\nti-B = antiserum against E. coli B. c Anti-0127 = antiserum against E. coli 0127.
HRBC HRBC-LPS H RBC-LPS HRBC-LPS
‘I’ x B
NMKI
HRBC HRBC-LPS HRBC-LPS
HRBC HRBC-LPS HRBC-LPS HRBC HRBC-LPS HRBC-LPS
HRBC HRBC-LPS HRBC-LPS HRBC H RBC-LPS HRBC-LPS
Antigen
‘I‘ X B
NXB
‘I‘ X B
N x B
TXB
Treatment
NMRI
NMRI
NMRI
Strain
3
EFFECT OF ANTIBODIES TO LPS FROM E. coli 0127 ON THE THYMUS-INDEPENDENCE
TABLE
THYMUS
INDEPENDENCY
OF RED
CELLS
423
tion may be responsible. Such an interpretation is compatible with the tolerance experiments, because it has been shown that tolerance induction to LPS results in an increased number of LPS-binding cells (5)) and the antibody transfer experiments. because antibodies would prevent LPS from interacting with the B cells. However, there is no evidence for a B-B cell interaction in the immune response. Therefore, this explanation remains as a theoretical, but so far unsupported, possibility. b. LPS is a conventional immunogen and is competent to induce specific antibody synthesis, as well as specific tolerance. However, it has been clearly demonstrated that LPS can also function as a nonspecific mitogen for B cells, but not for T cells (17, 18). Thus, LPS has the ability to stimulate DNA synthesis in B cells and it can also activate antibody producing cells to various noncross reacting antigens, as if specific antigen had been added (19). Finally, LPS can selectively stimulate IgM synthesis in vitro (20). The B cell mitogenic property of LPS may explain the present results. Thus, when LPS-coated red cells have reacted with B precursor cells having immunoglobulin receptors for the red cells, LPS will have the possibility to act as a mitogen for these cells and thereby activate them to division and differentiation, even in the absence of a T-B cell cooperation. The results of tolerance induction to LPS would also be compatible with this interpretation because the large amount of LPS injected will persist undegraded for a long time in the animals and can bind nonspecifically to many B cells. When these animals are injected with LPS-coated red cells, those B cells that recognize the red cells by immunoglobulin receptors are confronted with a specific antigenic st,imulus, by itself insufficient to activate the cells and, in addition, a large number of LPS molecules, contributed both by those presenb on the red cells and those previously injected. This would cause a stronger nonspecific stimulus to division than if the animals had not been pretreated. Passive transfer of antibody to LPS would decrease its ability to interact with the B lymphocytes and therefore this treatment would clecrease the capacity of LPS to stimulate the response to LPS-coated red cells. The second interpretation of the results cannot be formally proved, but is in agreement with the in vitro findings mentioned above, showing that LPS can activate B cells nonspecifically. It has also been demonstrated that, LPS can substitute for T cells in the induction of an immune response in vitro to red cell antigens (19). It follows that LPS can supplement for the T cell function, which helps activating B cells in the immune response. Therefore, the T-B cell cooperation may be nonspecific in nature, because the T cell signal to B cells may be substituted by a nonspecific B cell mitogen. Recent findings is in agreement with this postulate and demonstrate that at least one T cell signal to the B cells can be a nonspecific humoral factor, which by itself does not act as a mitogen, but in cooperation with an inducing agent (antigen or mitogen) can cause proliferation of B cells (21 1. The T cell independence of the immune response to LPS itself may be caused by the dual function of LPS, being both an antigen and a nonspecific mitogen. However, nonmitogenic antigens, such as PVP and pneunlococcal polysac&ari&s, are also T cell independent. Therefore, the B-B cell cooperation and the ability of these antigens to activate B cells directly by, e.g.. multiple bond formation still exists as plausible interpretations for their T cell independence.
424
MGLLER,
ANDERSSON,
AND
SJijBERG
ACKNOWLEDGMENTS The part of this work carried out in Sweden was supported by grants from the Swedish Medical Research Council, the Swedish Cancer Society, the Wallenberg Foundation and the Harald Jeansson’s Foundation. The technical assistance of Mrs. Kerstin Andersson is gratefully acknowledged. REFERENCES 1. Transplant.
Rev. 1, 1, 1969.
2. Miiller, G., and Michael, G., Cell. Ia2+nnxol. 2, 309, 1971. 3. Andersson, B., and Blomgren, H., Cell. I~mmtlzol. 2, 411, 1971. 4. Howard, J. G., Christie, G. H., Courtenay, B. M., Leuchars, E., and Davies, .4. J. S., Cell. Imnzunol. 2, 614, 1971. 5. Sjijberg, O., J. Exp. Med. 133, 1015, 1971. 6. Greaves, M. F., and Moller, E., Cell. Immunol. 1, 372, 1970. 7. Mijller, G., Nature (London) 207, 1166, 1963. 8. Moller, G., Cell. Imnmnol. 1, 573, 1970. 9. Westphal, O., Liiderlitz, O., and Bister, F., Z. Natwfors~h. 7, 148, 1952. 10. Britton, S., Immulzologv 16, 513, 1969. 11. Jerne, N. K., and Nordjn, A. A. S&ace 140, 405, 1963. 12. Mishell, R., and Dutton, R., J. Exp. Med. 123, 423, 1967. 13. Henry, C., and Jerne, N. K., J. Exp. Med. 126, 133, 1968. 14. Feldman, M., and Diener, E., J. Exp. Med. 131, 247, 1970. 15. McBride, R. A., and Schierman, L., J. Exp. Med. 131, 377, 1970. 16. Britton, S., Immulzology 16, 527, 1969. 17. Peavy, D. L., Adler, W. H., and Smith, R. T., J. Immunol. 105, 1453, 1970. 18. Andersson, J., Moller, G., and Sjgberg, O., Cell. Immzlnol. 3, 000, 1972. 19. Sjoberg, O., i2nderssoq J., and Moller, G., Europ. J. Imnwnology, In press. 20. .4ndersson, J,, Moller, G., and Sjoberg, O., Europ. J. Imnrzmology, In press. 21. Andersson, J., Miiller, G., and SjBberg, O., Eur. J. Immwology. In press.