Vol. 21 (11), 653-665, 1977
Induced Serum Factor
to of Antibody
Masao J. TANABE, Yoshiko TSURUMI, and Masayasu NAKANO
Department of Microbiology, Jichi Medical School, Tochigi
(Received for publication, April 25, 1977)
injected with LPS several hours in advance, contained some active substance capable
of enhancing anti-sheep red blood cell (SRBC) antibody responses in mice. Ac-
tivity of the sera was still retained after passage through a rabbit anti-LPS antibody-
coated Sepharose 4B column, but greatly reduced by passage through a rabbit anti-
mouse thymocyte antibody-coated Sepharose 4B column.
the sera was eluted through a Sephadex G-200 column at the same position as the
The addition of this substance to B cell rich spleen cell cultures in vitro in the
presence of SRBC generated tremendous numbers of antibody forming cells 4 days
after the incubation, suggesting that this substance was able to take over the helper
function of T cells in thymus dependent antibody responses. However, this sub-
stance was not capable of stimulating 3H-thymidine-uptake into cultured spleen cells.
The possible role of this substance in the adjuvant effect of LPS is discussed.
The sera obtained from blood of the mice, which had been intravenously
The active substance in
Lipopolysaccharide (LPS) obtained from Gram-negative bacteria has recently
become a focal point of interest in immunology because of its various effects on the
murine immune system. This substance itself has been characterized as a mitogen or
polyclonal activator for B lymphocytes (2, 9, 10, 27). On the other hand, when
LPS is injected with antigens concomitantly, prominent adjuvant effects on antibody
responses to these antigens are observed (3, 15). Mechanisms of the adjuvant effect
of LPS have still not been clarified, but there are two opposite views on the target
cells of LPS in lymphoid cells; i.e., the adjuvant effect of LPS results from (a) its
direct effect on antibody producing B lymphocytes (27), or (b) its stimulatory effect
on helper T lymphocytes participating in antibody response via some unknown
mechanism (3, 15). Effects of LPS on both antigen-stimulated B and T lymphocytes
may participate with the elicitation of its adjuvant effects on antibody responses in
We have already shown (29) that a mediator or soluble factor capable of stimu-
lating antibody response is produced in circulating blood after the injection of ad-
juvants or secondary antigenic stimulation, and this factor may play some role in the
enhancement of antibody responses. In this paper, we report on the evidence of the
M. J. TANABE ET AL
participation of this soluble factor in adjuvant effects of LPS in primary antibody
response against erythrocyte antigen in mice, and some characteristics of this factor.
MATERIALS AND METHODS
Mice. C57BL/6 mice of both sexes, 8-12 weeks of age from our own colony,
were used in all the experiments.
Antigen and immunization. Sheep red blood cells (SRBC) in Alsever's solution
were obtained commercially. Before use in vivo, these erythrocytes were washed three
times with physiological saline and resuspended in the saline (1 •~ 109 erythrocytes per
ml). Two-tenths ml each of the SRBC suspension (2 •~ 108 SRBC) was inoculated
intraperitoneally (ip) into mice. For in vitro experiments, SRBC were washed three
times with Eagle's minimal essential medium (MEM) and resuspended in MEM to
an appropriate concentration for inoculation into cultures.
Bacterial lipopolysaccharide (LPS). LPS was extracted from Salmonella typhimurium
LT2 with hot phenol-water according to the method of Westphal et al (34, 35) or with
cold n-butanol-water according to the method of Morrison et al (21). The LPS
extracted with hot phenol-water was purified by repeated centrifugation at 100,000 •~
to remove contaminating
The LPS extracted with
was with (20 ƒÊg/ml of pronase
45,000 Tryosin units/g, Kaken Chemicals, Co., Ltd., Tokyo, Japan) at 37 C overnight
to digest contaminating proteins and then this solution was concentrated by a rotary
evaporator (butanol LPS). For further purification, the butanol LPS solution (20-
40 mg/ml) was loaded onto Sepharose 4B columns (90 •~ 2.6 cm). LPS content in
each eluate was estimated by the assay of KDO (32). Butanol-LPS rich eluates
(tube numbers 24 to 29) were pooled, condensed by a rotary evaporator and stocked
in a refrigerator before use for experiments (column-passed butanol-LPS).
Preparation of anti-thymocyte-serum (ATS) and anti-LPS serum. Rabbit anti-mouse-
thymocyte-serum and anti-LPS-serum were obtained from blood of the rabbits 1
week after the last immunization, which had been intravenously (iv) immunized
repeatedly with either 5 •~ 108 C57BL/6 mouse thymocytes (5 times, 14 day intervals)
(6, 17) or 1010 heat killed organisms of S. typhimurium LT2 (7 times, 7 day intervals)
(22). These sera were inactivated at 56 C 30 min, and then adsorbed repeatedly
with one tenth volume of C57BL/6 mouse red blood cells until no hemagglutinin- or
hemolysin-titers to mouse erythrocytes could be detected in the sera.
Affinity column with ATS or anti-LPS serum. CNBr activated Sepharose 4B bead
gel (Pharmacia Fine Chemicals, Sweden) was swelled and washed by 1 mm HCl.
Anti-thymocyte- or anti-LPS-serum was coupled to the gel in 0.1 M NaHCO3-Na2-
CO3 buffer (pH 8.3, 0.5 M NaCl) for 2 hr at room temperature. The gels bound
around anti-thymocyte- or anti-LPS-antibodies were washed once with the same
buffer and then washed several times with acetate buffer (0.1 M NaCH3COO-CH3-
COOH pH 4, 0.5 M NaCl). Finally these gels were washed with phosphate buffered
saline (0.01 M K-PB pH 7.4, 0.15 M NaCl) and then packed into columns (1 •~ 10 cm)
SERUM FACTOR FOR ADJUVANT EFFECT OF LPS
" Endotoxin serum" and its chromatographic separation
. One hundred ƒÊg (in 0.2 ml
of saline) of column-passed butanol-LPS were injected intravenously (iv) into mice.
At indicated times after the injection, the blood of these mice were collected by car-
diac puncture. The sera were separated from the blood, pooled and inactivated at
C for 30 min.
Thus, the serum obtained from LPS-injected mice is termed
." To obtain an active substance from "endotoxin serum," the
serum was repeatedly chromatographed through a column (90 •~ 2.6 cm) of Sephadex
G-200 superfine at 4 C with 0.15 M phosphate buffered saline (PBS), pH 7.4, at a
pressure of 15 cm H2O (flow rate : 2.5 ml/hr).
Estimation of LPS content in "endotoxin serum." LPS content in the "endotoxin
serum" was estimated by the limulus test (36). Pregel solution, lysate of the ame-
bocyte of Limulus polyphemus (Teikokuzoki, Co., Ltd., Japan), was dissolved in 0.1 ml
of distilled water. One-tenth ml each of serially ten-fold diluted "endotoxin serum"
was dropped into the test tubes containing pregel solution and then the endpoint of
this serum in limulus gelation was determined after incubation at 37 C for 1 hour.
Spleen cell suspension and culture media for spleen cells. The spleen cell suspension was
prepared by teasing cells out of the splenic capsule in MEM containing with 10% of
FCS. After the elimination of undispersed cells by passage through fine nylon meshes
and placing the suspension in ice bath for 5 min, these cells were resuspended into
culture media. Nutrient mixture F-15 [Grand Island Biological Co., (Gibco),
Grand Island, N.Y.] supplemented with 10% heat-inactivated fetal calf serum was
employed as a routine culture medium to test for antibody responses. RPMI-1640
(Gibco) supplemented with 5% heat-inactivated human serum was employed as a
culture medium to test for the mitogenicity of spleen cells. If B cell rich suspensions
were desired for cultures, the spleen cells were treated for 45 min at 37 C with ade-
quately diluted ATS in the presence of fresh guinea pig complement (8) to kill T cells.
After this treatment, the cells were washed three times with fresh media and finally
suspended in the culture medium. The number of viable cells in the suspension was
calculated by the exclusion of trypan blue dye.
Assay for helper activity of "endotoxin serum" on antibody response. The cells were
cultured by the technique of Marbrook (18) with minor modifications. The spleen
cell suspension (1 •~ 107 viable cells in 0.2 ml of culture medium) was poured into the
inner compartment (10 •~ 100 mm) of the culture vessel, and then 0.2 ml of SRBC
with or without
ml of chromatographic eluates of the
added to same
. The final volume of the
culture in the inner compartment was increased to one ml by the addition of culture
medium. Glass vial bottles for scintillation counting (Packard Instruments) were
used as the outer compartment for the nutrient culture medium and 15 ml of the
medium was poured into the outer compartment. Both the inner and outer com-
partments were separated by a visking membrane, but small molecular substances
could pass freely through the membrane. The cells were cultured at 37 C for 4 days
in an atmosphere of 95% air and 5% CO2. At the end of the cultivation, the num-
ber of plaque forming cells (PFC) against SRBC in the culture was determined by
Jerne's method (16). As a rule, duplicate tubes were used for the determination of
M. J. TANABE ET AL
in one group,
The effect of "endotoxin
and the means
of the PFC-
Assay for mitogenicity of "endotoxin serum." Two-tenths ml of spleen cell sus-
pensions (1 •~ 107 nucleated cells per ml of RPMI-1640 medium supplemented with
5% human serum) and 0.1 ml of chromatographic eluates of "endotoxin serum" or
of culture medium were poured into a disposable tissue culture tube (12 •~ 75 mm)
(Falcon Plastics, No. 2058, Los Angeles, Calif.). These cell suspensions were finally
adjusted to a one ml volume with the addition of culture medium and then cultured
at 37 C in an atmosphere of 5% CO2 in humidified air for 72 hr. The cultures
received 1 ,uCi of 3H-thymidine (thymidine-methyl-T, 11.6 Ci/m mole, Daiichi Pure
Chemicals, Co., Ltd., Tokyo, Japan) twenty hours before harvesting. Incorporation
of labeled 3H-thymidine into the acid insoluble precipitate was measured by the
standard method (1).
Assay for antibody response against SRBC in vivo. Immune response of mice to
SRBC was determined by counting the numbers of 19S plaque-forming cells (PFC)
in their spleens according to the direct technique of Jerne et al (16) 3 days after im-
munization. All the PFC values were expressed as the numbers of PFC per in-
dividual spleen. As a rule, five mice were used for the determination of PFC re-
sponse in one group, and the mean of the PFC for the five mice and its standard
error were calculated.
Preparation of Homogeneous and Macromolecular LPS
Butanol-LPS and phenol-LPS were loaded onto Sepharose 4B columns, and the
chromatographic profiles obtained are shown in Fig. 1. Butanol-LPS gave one
prominent peak while phenol-LPS gave two sluggish peaks. The peak fractions of
butanol-LPS (tube numbers from 24 to 29) were collected, pooled together and con-
densed by a vacuum evaporator. This preparation should contain homogeneous
and macromolecular LPS (approximately M.W. 1.5 •~ 106). Therefore, we used
this column-passed butanol-LPS for the following experiments.
Adjuvant Effect of "Endotoxin Serum" on Antibody Response
Mice were iv injected with column-passed butanol-LPS (100 ,ug in 0.2 ml of
saline) and the blood of these mice was collected 1, 2, 3, 4, and 5 hr after the injection.
The sera were separated from the blood, and termed "endotoxin serum" after
1, 2, 3, 4 or 5 hr. Then, these sera were injected ip with 2•~ 108 SRBC into mice.
PFC responses in the spleen of these mice were determined 3 days after the injection
(Table 1). When the mice were injected with the antigen and "endotoxin serum"
after 1 to 4 hr simultaneously, these mice showed greater numbers (about triple) of
anti-SRBC 19S PFC in their spleens than immune controls. The "endotoxin serum"
after 5 hr still has a significant enhancing effect on PFC response although the extent
SERUM FACTOR FOR ADJUVANT EFFECT OF LPS
of this effect seems to become weaker. These results indicate that "endotoxin serum"
has some adjuvant effects on anti-SRBC PFC responses in the spleen of mice.
Fig. 1. Chromatographic profiles of butanol-LPS and phenol-LPS through Sepharose 4B
LPS (100-200 mg in 5 ml) was loaded onto a column (90 •~ 2.6 cm) of Sepharose 4B and
eluted at 4 C with 0.15 M PBS, pH 7.4, at a pressure of 20 to 30 cm H2O (flow rate: 6 ml/
hr). Five ml each of eluate was collected in tubes and a 0.1 ml aliquot from each tube was
subjected to an assay for LPS content. The relative content of LPS in eluates was photo-
metrically determined by absorbance at 552 nm after the coloring of KDO with thio-
barbituric acid (12). 0, butanol-LPS; e, phenol-LPS.
Fi g. 2.
Chrom atographi c(● )and
rechrom atographi c(□ , ○ )profi l es of, "endotoxi n serum , ,
serum" (2 ml)
was loaded onto a column (90 •~ 2.6 cm) of superfine Sephadex
G-200 and chromatographed at 4 C with 0.15 M PBS, pH 7.4, at a pressure of 15 cm H20
rate; 2.5 ml/hr).
of each of the
in tubes and
29 in elution absorbance nm. Eluates in to 40
and 41 to 60 were pooled respectively, condensed and again rechromatographed through
superfine Sephadex G-200 under the same conditions mentioned above.
Table 1. Adjuvant effect of "endotoxin serum"
M. J. TANABE ET AL
Active Substance in "Endotoxin Serum"
A substantial amount
of LPS may still remain
pre "endotoxin serum" from blood been
viously with 100 ƒÊg of LPS. Therefore, the amount of LPS contained in "endotoxin
serum" should be checked. "Endotoxin sera" after 3 or 4 hrs were serially diluted.
tenfold and then these diluents were mixed with limulus amebocytes. The end-
points of these sera for obtaining positive gelation were •~ 102 or •~ 100 respectively
(the lower part of Table 2). The minimal amount of LPS which caused gelation in
serum was also examined. The control sera containing 10-3 ƒÊg/0.1 ml or more
LPS gave positive gelation (the upper part of Table 2). Thus, these "endotoxin
sera" after 3 and 4 hours should contain about 10-1 and 10-3 ƒÊg/0.1 ml of LPS
respectively. Usually, an injection of 10-1 ƒÊg or less of butanol-LPS together with
SRBC causes hardly any adjuvant effect on the anti-SRBC PFC response in the
spleen of mice (data not shown). Therefore, the adjuvant effect of the "endotoxin
serum" should not be attributed to the effect of LPS. These results suggest that
injection of LPS into mice may produce or release some active substance capable
of enhancing antibody response into the circulating blood.
Chromatographic Purification of Active Subtances in "Endotoxin Serum"
To purify the active substance in "endotoxin serum" and estimate its molecular
weight, "endotoxin serum" was loaded onto a column of superfine Sephadex G-200.
The elution profiles of "endotoxin serum" on this chromatogram are shown in Fig. 2.
There were 3 peaks of IgM, IgG and albumin respectively. The eluates making up
the first and second peaks (tubes 29 to 40) were pooled together and the other eluates
forming the third peak (tubes 41 to 60) were also pooled. These pooled eluates were
condensed by an evaporator and then again loaded into the column.
graphic profiles of these pooled eluates one also shown by the lump in Fig. 2. The
tests of column-passed
(A) Limulus test of column-passed butanol-LPS
(B) Limulus test of "endotoxin serum"
SERUM FACTOR FOR ADJUVANT EFFECT OF LPS
rechromatographic eluates of IgM and IgG fractions (tubes 29 to 38) and of the
albumin fraction (tubes 41 to 49) were again pooled and condensed to the initial
volume before using. These solutions were termed "fraction 1 (F-1) and 2 (F-2)."
Mice were ip injected with 0.3 ml of either F-1 or F-2, together with 108 SRBC.
The adjuvant effect of these fractions on PFC responses of spleens were estimated 3
days after the injection. As shown in Table 3, the mice injected with F-2 produced
about 9 times greater PFC in their spleens than the controls. This fact indicates
that the active substance in "endotoxin serum" for the adjuvant effect on PFC
responses is present in F-2, but not in F-1, and the molecular weight of this substance
may be similar to that of mouse albumin (M.W. 6-7 •~ 104). The molecular weight
of butanol-LPS was quite large (M.W. 1.5 •~ 106, see Fig. 1). Therefore, this finding
seems to support the view that the adjuvant effect of "endotoxin serum" is not due to
the effect of LPS contaminants.
Helper Activity of the Active Substance in Anti-SRBC Antibody Synthesis
This active substance in "endotoxin serum" is capable of helping antibody pro-
duction of B lymphocytes in vitro. In other words, the function of T lymphocytes in
antibody response against thymus-dependent erythrocyte antigen can be replaced
by this active substance. B cell rich spleen cells were cultured for 4 days in vitro in
the presence of SRBC with or without F-2. As shown in Table 4, the addition of
either 40- or 160-fold diluent of F-2 generated tremendous numbers of PFC in the
Table 3. Adjuvant effect of fraction 1 and fraction
2 prepared from "endotoxin serum"
Table 4. Helper activity of F-2 of "endotoxin serum" on antibody
response of cultured B lymphocytes")
M. J. TANABE ET AL
antibody responses against thymus-dependent antigens (27). However, the action
of LPS in our experimental system was poor since the culture added to LPS instead
of F-2 produced only double the numbers of PFC than controls. The finding that
F-2 has stronger functions as a helper in antibody production of B cells than LPS
itself also supports the possibility that the greater part of adjuvant action occurs via
some soluble factor induced by LPS.
LPS is known to be able to substitute for the helper function of T cellsin
No Mitogenicity of Franction-2 for Cultured Lymphocytes
It is well known that LPS has some mitogenic effects on B cells and initiates
de novo DNA synthesis in them (9). If the F-2 obtained from "endotoxin serum"
contains a small amount of fragmented LPS and the fragmented LPS still retains
endotoxic activity, this fraction should be capable of stimulating DNA synthesis of
cultured spleen cells. Thus, the uptake of 3H-thymidine into the cultured spleen
cells with or without the addition of F-2 was examined. Spleen cell suspension
(2 •~ 106 spleen cells in 1 ml per tube) was incubated for 72 hr in the presence of F-2
(diluted from 10 to 2,560 fold) or of some butanol-LPS (2 to 200 ƒÊg) or phenol-LPS
(2ƒÊg). Both butanol-LPS and phenol-LPS had great enhancing effects on the in-
corporation of 3H-thymidine into the cultured cells. However, the addition of F-2
did not elicit any significant enhancement of 3H-thymidine uptake into the cultured
cells (Table 5).
Absorption of Adjuvant Activity of "Endotoxin Serum" by Anti-LPS- or Anti- Thymocyte-Serum
Binding around Sepharose 4B Beads
The following investigations were carried out to determine whether or not this
active substance in the "endotoxin serum" originated from lymphocytes. Rabbit
anti-LPS-serum or rabbit anti-mouse-thymocyte-serum was bound to the surfaces of
Table 5. 3H-thymidine uptake of cultured spleen cells in the
presence of F-2 of "endotoxin serum" or LPS
SERUM FACTOR FOR ADJUVANT EFFECT OF LPS
cyanogen bromide activated Sepharose 4B beads. "Endotoxin serum," which had
been collected from the blood of mice 4 hr after LPS-injection, was loaded onto
these columns and eluted with PBS at 4 C. After the elution, the eluates were
collected and condensed to the initial volume by using a collodion bag. These
column-passed sera were injected together with SRBC into mice and 3 day slater the
adjuvant effects of these sera were estimated by anti-SRBC PFC response in the
spleens of these mice. As shown in Table 6, the serum which had been passed
through the anti-LPS-serum-coated column retained about 88% activity, while the
serum passed through the ATS-coated column showed only 19% activity.
that the adjuvant activity of "endotoxin serum" is absorbed with the ATS-coated
beads suggests that this active substance may be the product of lymphocytes.
In a previous paper (29), we suggested that mice injected with LPS produce
some mediator which enhances antibody response in their blood, and this mediator
might play some important role in augmenting antibody responses by adjuvants or
the second injections of specific antigen. In the present study, we attempted to
obtain some active substance from mice injected with LPS and clarify the mode of
action of this substance.
The "endotoxin serum" obtained from the blood of the mice, which had been
injected with 100ƒÊg of LPS several hours in advance, contained some adjuvant
substance capable of enhancing the anti-SRBC antibody response of mice (Table 1).
One may claim that the adjuvant effect of the serum is not due to some special sub-
stance, but to the LPS contaminating it. However, the results obtained by the
limulus gelation test indicate that the serum collected 4 hours after the LPS-injection
does not contain enough LPS to cause on adjuvant effect (Table 2). Therefore, the
adjuvant effect of "endotoxin serum" should not arise from LPS but from another
unknown substance. There is other evidence to distinguish the active substance in
Table 6. Adjuvant activities of "endotoxin serum" before and after
passage through anti-LPS-serum or anti-thymocyte-serum
(ATS) binding Sepharose 4B column
M.J. TANABE ET AL
"endotoxin serum" from contaminating LPS. 1. Adjuvant activity of "endotoxin
serum" could not be decreased by passage through an anti-LPS antibody-coated
Sepharose 4B column, but it was lost significantly through an ATS-coated column
(Table 6). 2. The molecular weight of LPS used in these experiments was about
1,500,000 daltons, while the active substance was eluted through the column at a
similar position as that of mouse albumin (Figs. 1, 2), suggesting that the molecular
weight of this substance was significantly smaller than that of LPS. 3. LPS has
mitogenic activity for B lymphocytes, while the active substance in "endotoxin serum"
has no mitogenicity (Table 5). Thus, these results suggest that injection of LPS
into mice produces some substance (mediator) enhancing antibody response, and
the "endotoxin serum" contains this active substance.
cological mediator was not clarified in our experiments.
the release of DNA in circulating blood a few hours after the injection of LPS. DNA
has an adjuvant effect on anti-SRBC antibody response in mice (7). This DNA,
which had been released from cells damaged by endotoxic LPS, might be one of
the candidates for the active substance in "endotoxin serum."
that the DNA does not become attached to the fragmented lymphoid cell membrane,]
the finding that adjuvant activity of "endotoxin serum" is diminished by passage
through an ATS-coated column (Table 6), seems not to support the idea that DNA
is a major substance causing adjuvant activity in "endotoxin serum."
Unanue et al (31) reported that Listeria-infected or LPS-stimulated macrophages
produced a mediator capable of stimulating antibody response. However, their
mediator seems to differ from ours because their mediator has mitogenic activity for
lymphocytes. Our substance may have some immunologically specific markers
related to lymphoid cell surfaces since it can combine with anti-thymocyte antibodies.
If this supposition is correct, this substance probably comes from lymphocytes or
lymphoid-organs. In the preliminary experiments (data not shown), we obtained a
pharmacologically active adjuvant substance from the supernatant of T cell cultures
but not of B cell cultures, in the presence of LPS in vitro. Thus, the effective sub-
stance contained in "endotoxin serum" may be one of the pharmacological mediators
derived from T cells.
The origin of this pharma-
Fournie et al (14) reported
LPS is mitogenic on B cells but not T cells. Therefore, the action of LPS on T
lymphocytes tends to be overlooked. Recently, there have been several reports that
T cells may be stimulated by LPS, either directly or indirectly. Scheid et al (25)
have demonstrated that when spleen cells obtained from unde mice are cultured with
LPS, these cells have ƒÆand TL antigens on their surfaces which have never been
observed in the spleen cells of these mice. Their data suggest that LPS can perhaps
differentiate precursors in the spleen cells of nude mice from matured T lymphocytes
with TL antigens. A startling result related to activity of LPS was shown by Forbes
et al (13). They found that when thymocytes of mice were cultured with LPS and
minimally mitogenic doses of concanavalin A or submitogenic numbers of mitomycin-
treated allogeneic spleen cells, de novo synthesis of DNA in these cells was significantly
greater than that of controls without LPS. These data indicate that LPS is capable
of acting in some way to augment synergistically T cell proliferation by concanavalin
SERUM FACTOR FOR ADJUVANT EFFECT OF LPS
A or allogeneic cells. Furthermore, Ritter et al. (24) reported that LPS enhanced
the generation of cytotoxic T cells. These examples demonstrate that LPS has some
ability to enhance T cell functions although it does not have any mitogenic effects
on T cells.
Some pharmacologically active mediators possessing the helper function were
found by several investigators. One of these mediators is antigen-specific because it
is obtained from antigen-stimulated T cells and acts on B cells in the presence of the
same antigen (12, 30). Another was antigen-nonspecific since the mediator was
produced by a GVH reaction in vitro (4, 11), or activation of T cells with mitogens
(19, 26). T cells perhaps have some receptor for LPS as in the case of B cells or
erythrocytes (28, 33). Attachment of LPS to the receptors on T cells may trigger
them to produce a nonspecific mediator.
differ from that of T cell mitogens or the GVH reaction since LPS does not have any
mitogenic effects on T cells. Furthermore, McIntire (20) reported that the adjuvant
effect of LPS was separable from its mitogenic effect on B lymphocytes. These
findings suggest that the adjuvant effects of LPS may be intimately connected with
other mechanisms rather than polyclonal B cell activation by LPS.
This action of LPS on T cells seems to
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Requests for reprints should be addressed to Dr. Masao J. Tanabe, Department of Micro-
biology, Jichi Medical School, Tochigi-ken 329-04, Japan.