By
§
From the * Department of Laboratory Medicine and Pathology, Department of Internal Medicine, and § Howard Hughes Medical Institute and Center for Immunology, Washington University School of
Medicine, St. Louis, Missouri 63110
LT-deficient (LT
/
) mice show altered splenic microarchitecture. This includes loss of
normal B cell-T cell compartmentalization, of follicular dendritic cell (FDC) clusters, and of
ability to form germinal centers (GC). LT
/
mice immunized with sheep red blood cells
(SRBC) produced high levels of antigen-specific IgM but no IgG in either primary or secondary responses, demonstrating failure of Ig class switching. This inability to switch to IgG could
have been due to the altered splenic microarchitecture in these mice. Alternatively, it could have been due directly to a requirement for LT
expression by lymphocytes cooperating in the
antibody response. To investigate this, we performed reciprocal spleen cell transfers. When irradiated LT
/
mice were reconstituted with wild-type splenocytes and immunized immediately with SRBC, splenic microarchitecture remained disturbed and there was no IgG response. In contrast, when irradiated wild-type animals received splenocytes from LT
/
mice,
follicle structure and a strong IgG response were retained. These data indicate that LT
-deficient B cells and T cells have no intrinsic defect in ability to generate an IgG response. Rather, the altered microenvironment characteristic of LT
/
mice appears to result in impaired ability to switch to a productive IgG response. To investigate whether prolonged expression of
LT
could alter the structure and function of spleen follicles, reciprocal bone marrow (BM)
transplantation was performed. Six weeks after reconstitution of LT
/
mice with wild-type BM,
spleen follicle structure was partially restored, with return of FDC clusters and GC. B cell/T
cell compartmentalization remained abnormal and white pulp zones were small. This was accompanied by restoration of IgG response to SRBC. Reconstitution of wild-type mice with
LT
/
BM resulted in loss of FDC clusters and GC, and loss of the IgG response, although
compartmentalized B cell and T cell zones were largely retained. Thus, defective IgG production is not absolutely associated with abnormal B cell and T cell compartmentalization. Rather,
expression of LT
supports the maturation of spleen follicle structure, including the development and maintenance of FDC clusters, which supports Ig class switching and an effective IgG
response.
Lymphotoxin- LT LT Mice.
C57BL/6J and 129Sv mice were obtained from The
Jackson Laboratory (Bar Harbor, ME). LT Measurement of Antigen-specific Ig.
Specific antibodies were measured and analyzed as previously described (15). In brief, Immulon 4 plates (Dynatech Laboratories, Inc., Chantilly, VA) were
coated with SRBC (150 µl at 5 × 107/ml) suspended in 0.25%
glutaraldehyde in PBS. Diluted mouse sera were then added and
incubated at 4°C for 1 h. Alkaline phosphatase-conjugated goat
anti-mouse isotype-specific antisera (Southern Biotechnology, Birmingham, AL) were diluted 1:500 for IgM and 1:2,000 for total IgG or IgG subclasses, and 100 µl were added and incubated at 4°C for 1 h, followed by washing and addition of the alkaline phosphatase substrate p-nitrophenyl phosphate (Sigma Chem.
Co., St. Louis, MO) at 1 mg/ml. The mean OD at 405 nm from
triplicate wells was compared to a standard curve of titrated serum
to calculate the relative units (RU) using linear regression analysis
(15). The results represent mean ± SEM.
Transfer of Splenocytes.
Whole spleen cell suspensions were
prepared from single mouse donors by mincing the spleen with
scissors and teasing the tissue fragments between two frosted microscope slides. Recipients were prepared by irradiation with 750 rads 3 h before cell transfer. As indicated, SRBC were mixed together with the spleen cell suspensions before i.v. injection. Each
recipient received all of the cells derived from a single donor
spleen.
BM Transplantation.
BM was harvested and recipients were
prepared as described previously (16). Recipient mice were lethally irradiated with 1,050 rad (10.5 Gy) and reconstituted with
5 × 106 donor BM cells. 6 wk after transplantation, recipients
were immunized i.p. with 108 SRBC and serum samples were
collected 10 d after primary or secondary immunization. 6 mo after transfer, wild-type mice reconstituted with LT Evaluation of Spleen Follicle Structure.
Spleens were harvested,
embedded in O.C.T. compound (Miles, Elkhart, IN), and frozen
in liquid nitrogen. Frozen sections (6-10 µm thick) were fixed in
cold acetone. Endogenous peroxidase was quenched with 0.2%
H2O2 in methanol. After washing, the sections were stained by
first incubating with FITC-conjugated B220 (PharMingen, San
Diego, CA), and biotinylated anti-CR1 8C12 or Thy1.2 (PharMingen) or PNA (Vector, Burlingame, CA), all at 1:100 dilution.
Horseradish peroxidase (HRP)-conjugated rabbit anti-FITC (Dako,
Glostrup, Denmark; diluted 1:10) was added 1 h later. Sections
were then incubated for 1 h with one drop of alkaline phosphatase (AP)-conjugated streptavidin (Zymed, South San Francisco, CA) and color development for bound AP and HRP was
with an AP reaction kit (Vector) and with diaminobenzidine.
Previous studies
of LT When wild-type mice were immunized by injection of
107 SRBC into the footpad, they generated a robust IgG
anti-SRBC response (Fig. 1 a). In contrast, when LT
LT
The reduced IgG response in LT LT Wild-type mice that were irradiated and immunized
with SRBC without reconstitution with splenocytes showed
no detectable B220+ or Thy-1+ cells in their spleens and
produced no detectable anti-SRBC IgM or IgG at day 10 (data not shown), indicating that development of an antibody response could not be supported directly by any radioresistant cells in the recipient animals under these experimental conditions. When irradiated wild-type mice were
treated with LT
Interestingly, although the structure of the spleen white
pulp nodule and the ability to mount an antigen-specific
IgG response are disturbed in LT It is thought that establishment of proper B cell-T cell interactions in lymphoid follicles is essential for the initiation and maturation of humoral
responses to T cell-dependent antigens. Within follicles,
clustered follicular dendritic cells (FDC) with complement
receptors 1 and 2 and FcR are known to trap immune complexes and are thought to support development of GC
in which the development of effective IgG responses occurs (9, 15). In a previous study, we demonstrated that
LT To study the relationship between altered splenic microarchitecture and the ability to generate an anti-SRBC
IgG response, mice that had been irradiated and treated
with infusions of spleen cells were analyzed histologically.
Wild-type mice reconstituted with either wild-type or
LT
In this short-term spleen cell reconstitution model, LT BM transfer provides an alternate model
to evaluate the role of LT
Our data are in contrast to those of Muller et al. (17).
These investigators studying mice with functional ablation
of both the LT To test the impact of BM reconstitution and its accompanying restoration of FDC clusters and GC reactivity on
the character of the antibody response, we immunized
mice 6 wk after BM transplantation and analyzed antigenspecific serum IgG in both the primary and the secondary
responses. LT
Further support for the role of FDC clusters in the development of a robust IgG response comes from analysis of
the spleen histology and anti-SRBC IgG responses over
time after spleen cell transfer. In irradiated wild-type mice
that had received LT One difficulty in interpreting the results of longer term
spleen cell transfer experiments is that recipient lymphoid
cells can reconstitute the animals and compete with the
transferred cells. We have, therefore, performed similar experiments transferring wild-type or LT The data reported here demonstrate that the IgG response of LT Although these preliminary studies do not address the
mechanism of the adjuvant effect, they demonstrate strong
correlation between the presence of FDC clusters and GC
responsiveness for effective isotype switching. The adjuvant-induced restoration of anti-SRBC in LT In conclusion, the studies presented here demonstrate
that LT Additional studies of the TNF family members OX40
and OX40L have shown that interactions between this
ligand-receptor pair are required for the formation of antibody forming cell foci, but not for the formation of morphologically intact GC (20). Furthermore, signaling through
the nerve growth factor receptor, another member of the
TNFR superfamily, provides autocrine stimulation of memory B cells (21). Thus, signaling via multiple members of
the TNF ligand-receptor family supports efficient maturation of the B cell response, each probably acting at a different activation stage. It is well established that certain members of this family are capable of regulating the expression
of others (22, 23). It remains to be determined whether
members of this family act sequentially or via independent
pathways to support the maturation of the antibody response.
(LT
)1 shares structural features with
the related cytokine, TNF
. Both LT
and TNF
exist in solution as homotrimeric proteins. In these forms,
they share biological activities by virtue of their similar
binding to the two defined TNF receptors, TNFR-I, and
TNFR-II. Signaling via these two receptors modulates a
wide variety of immune and inflammatory responses (1, 2). LT
also exists in a heteromeric form with the type II
membrane protein LT
, which in its most prevalent form
on the membrane has the stoichiometry LT
1LT
2. The
LT
1LT
2 heteromer has no measurable affinity for TNFR-I
or TNFR-II, but does interact with high affinity with the
TNFR-related protein (also designated the LT
receptor, or
LT
R) (3).
/
mice are born with defective development of
LN and Peyer's patches (PP) (6). In LT
/
mice, spleen
structure is also disturbed, with small white pulp follicles
that fail to segregate T cell and B cell zones, and fail to generate clusters of FDC or GC (6). Proper spleen microarchitecture, including the presence of primary and secondary lymphoid follicles that contain FDC, is thought to be
required for all features of a mature T cell-dependent B cell
response, including Ig class switching, affinity maturation,
and development of antibody secreting cells (9). Consequently, it was striking that LT
/
mice were able to generate high affinity anti-NP IgG antibody after immunization
with high doses of the T cell-dependent antigen 4-hydroxy3-nitrophenyl-ovalbumin (NP-OVA) adsorbed to alum
(8). In contrast, there was impaired production of high affinity anti-NP IgG when LT
/
mice were immunized
with low doses of NP-OVA absorbed to alum. Banks et al.
(12) using an independently derived LT
/
mouse strain,
have shown impaired IgG responses in LT
/
mice after
subcutaneous immunization with KLH absorbed to alum or immunization with viral antigens. Further studies examining mice deficient in both TNF
and LT
demonstrated
variable IgG responses, with deficient IgG responses after
intraperitoneal immunization with SRBC but retained responses against vesicular stomatitis virus after an infectious
challenge (13). Recently prepared TNF
/
mice (14) are
reported also to have an impaired IgG anti-SRBC response
but a strong response to DNP-KLH adsorbed to alum.
Thus, the role of LT
in supporting an effective IgG response to SRBC remains incompletely defined.
/
mice manifest a complex phenotype that includes an absence of LT
expression and also established
abnormalities of lymphoid tissue development and structure (6). The present study was undertaken to investigate
the requirements for LT
expression in lymphoid cells and
for intact lymphoid tissue structure to support production
of an IgG responses to the T cell-dependent antigen
SRBC. We report that LT
/
mice responded with high
levels of IgM but very low levels of IgG after immunization
with SRBC in the absence of adjuvant. Experiments in
which suspensions of mature spleen cells or of T cell-depleted bone marrow were transferred to wild-type or LT
/
mice demonstrated that certain elements of spleen follicle
structure are plastic and are determined by the presence of
LT
-expressing cells. These experiments also demonstrated
that LT
/
B cells and T cells in a structurally intact lymphoid tissue environment are competent to perform Ig isotype switching. In contrast, disturbed lymphoid tissue structure
caused by absence of LT
and manifested by absence of
clusters of FDC is associated with an inability to form an
effective IgG response. Thus, LT
produced by bone marrow (BM)-derived cells establishes a permissive environment for an effective IgG response.
/
mice (6) were
maintained on a mixed 129Sv × C57BL/6 background and were
bred under specific pathogen-free conditions.
/
BM
showed retention of LN and PP, while LT
/
mice reconstituted with wild-type BM showed no evidence of de novo LN or
PP development.
LT/
Mice Immunized with SRBC Produce High Levels
of Antigen-specific IgM but Low Levels of IgG.
/
mice (8, 12) have showed variable IgG responses
after immunization with T cell-dependent antigens. It has
been suggested that this variability was due to difference in
the immunization protocols, in the routes of immunization, and in the nature of the antigens used. SRBC have
been commonly used for studies of antibody responsiveness
because they are T cell-dependent antigens, do not require
adjuvant to elicit a strong response, and can elicit IgG antibodies following immunization by either the intradermal,
subcutaneous, i.p., or i.v. routes. Studies by Eugster et al.
(13) using mice deficient in both LT
and TNF
demonstrated that isotype switching in these mice was defective
after immunization i.p. with SRBC. This indicated that either TNF
or LT
or both play an important role in the immune response against this antigen. These investigators
subsequently showed that transfer of wild-type BM to these
LT
/
/TNF
/
mice restored IgG responsiveness to this
antigen (17). This indicated that LT
and/or TNF
-
expressing cells transferred in BM determined IgG responsiveness; however, it did not define the mechanisms of action of the cytokines. Furthermore, the recent report that
TNF
is required for effective isotype switching to SRBC
(14) underscores the fact that the selective role of LT
in
IgG responses is not defined. The following studies were
performed to define the role of LT
in IgG production and
to dissect the mechanisms of LT
's effects in response to
SRBC.
/
mice were similarly immunized, they showed no detectable
anti-SRBC IgG after either the primary immunization or
secondary boost. This clearly indicated that, in addition to
TNF
, LT
is required for an intact IgG response against
SRBC.
Fig. 1.
Anti-SRBC IgG responses after subcutaneous or intraperitoneal immunization. Groups of 3-5 mice (8-wk-old) (wild-type, filled symbols; LT/
, open symbols) were immunized at day 0 in the footpad (A,
107 SRBC ) or by intraperitoneal injection (B, 108 SRBC) and given a
boost with the same dose on day 21. SRBC-specific IgG was measured
using an ELISA. Data shown represent the means ± SEM of triplicate determinations from 3-5 mice. RU, relative units. One representative experiment of three is shown.
[View Larger Version of this Image (19K GIF file)]
/
mice are born with defective development of
LN and PP and with altered splenic microarchitecture. LN
are considered to be important structures for collecting and
concentrating antigens. We considered that the lack of LN
in LT
/
mice might account for their impaired IgG response after peripheral immunization; however, immunization of LT
/
mice with higher doses of SRBC (1 × 108
or 6 × 108) i.p. still result in no primary antigen-specific
IgG response and a markedly reduced response after a secondary boost (Fig. 1 b). Even when mice were immunized
with a high dose of SRBC (1 × 108) i.v. to bypass the LN
and to deliver antigen directly to the spleen, the primary
IgG response of LT
/
mice was only at the lowest limit
of detection in the assay (Fig. 2 a), <1% that of wild-type
animals. After an i.v. boost, a small IgG anti-SRBC response was detected, but again at a level <3% that seen in
wild-type mice.
Fig. 2.
Anti-SRBC IgM and IgG responses in LT/
mice after intravenous immunization. Groups of 3-5 mice (8-wk-old) were immunized i.v. with 108 SRBC in PBS and boosted with the same dose 21 d
later. Serum anti-SRBC IgM (A) and IgG (B) were measured by ELISA.
Symbols and RU are as described in Fig. 1.
[View Larger Version of this Image (24K GIF file)]
/
mice did not simply represent a delay, as anti-SRBC IgG remained low 2-3
wk after both the initial immunization and after the secondary boost. Measurement of the levels of anti-SRBC
IgM, however, showed the presence of a brisk anti-SRBC
B cell response (Fig. 2 b). In fact, compared to wild-type
mice, LT
/
mice showed prolonged persistence of the
IgM response and a dramatically increased IgM response after a secondary boost. Thus, the reduced IgG production in
i.v. immunized LT
/
mice did not represent failure to
deliver sufficient antigen to activate the responding B cell
pool, or failure to activate these antigen-specific cells, but
rather it represented a failure of effective isotype class
switch or of affinity maturation to produce anti-SRBC IgG
with sufficient affinity for efficient detection.
-expressing Splenocytes Are not Sufficient to Direct IgG
Production in LT
/
Mice.
/
mice show altered splenic
microarchitecture without distinct B cell and T cell zones
and with absence of FDC and GC (6). Any of these
structural changes could interfere with the development of
a successful IgG response. Alternatively, LT
itself expressed by mature B and T lymphocytes could be an essential factor for the lymphocyte activation which is required
for an effective isotype switched response against SRBC.
To test whether intact splenic microarchitecture or LT
expressed by lymphocytes are required to activate Ig isotype switching, we performed reciprocal transfers of splenocytes. In these experiments, we either introduced wildtype LT
expressing B cells and T cells into the abnormal
structural environment of LT
/
mice, or we introduced
LT
-deficient cells into the normal structural environment
of wild-type mice.
/
splenocytes and were immunized i.v.
with SRBC, they produced high levels of IgG anti-SRBC
similar to wild-type mice that had received wild-type splenocytes (Fig. 3). This indicated that LT
-deficient B cells
and T cells have no intrinsic defect in Ig isotype switching, and that LT-expressing lymphocytes are not required for
effective anti-SRBC IgG production in the context of an
LT wild-type environment. In contrast, when LT
/
mice were irradiated and reconstituted with wild-type splenocytes, they failed to mount a detectable IgG response to
i.v. SRBC (Fig. 3). Thus, mature LT
-expressing lymphocytes are not sufficient for IgG isotype switching. Rather,
underlying wild-type spleen microarchitecture or spleen
stromal and non-lymphoid cellular elements which rely on
LT
for their proper formation may be necessary for the
IgG response against SRBC.
Fig. 3.
IgG production after
spleen cell transfer. Wild-type or
LT/
mice were irradiated
with 750 rad, then treated with
an infusion of spleen cells from
wild-type or LT
/
donors together with 108 SRBC. Ten days
later, serum was collected and
anti-SRBC IgG was measured
by ELISA. The results represent
mean ± SEM of 3-5 mice per
group. This experiment was repeated five times with similar results.
[View Larger Version of this Image (24K GIF file)]
/
mice, several other
features of normal immune responsiveness appear to be
fully retained. First, splenocytes harvested from either wildtype or LT
/
mice that had been immunized i.p. with
108 SRBC showed indistinguishable proliferative responses
when challenged in vitro with SRBC (data not shown).
Similarly, when wild-type or LT
/
mice were sensitized
with trimethlyamine in a hapten-specific delayed type hypersensitivity response and were challenged 7 d later by injection of hapten into the hind footpad, similar amounts of
footpad swelling were observed (data not shown). Finally,
LT
/
and wild-type mice rejected H-2 disparate skin allografts in similar fashion, with mean graft survival of 11 ± 1.3 d in wild-type recipients and 13 ± 1.3 d in LT
/
recipients. All of these data suggest that T cell function is retained in the LT
/
mice. Furthermore, since dendritic
cells are thought to act importantly during the activation of
these T cell-dependent responses, they suggest that dendritic cell function is retained in spite of the disturbance in
spleen structure.
/
mice show altered B cell-T cell zones, loss of FDC
clusters and absence of immune complex trapping (6, 8).
/
splenocytes showed similarly segregated B cell and
T cell zones (Fig. 4, A and C) and clusters of FDC (Fig. 4,
E and G). This was associated with competence for antiSRBC IgG responses (see Fig. 3). In contrast, when irradiated LT
/
mice were reconstituted with either wild-type
or LT
/
cells, B cell and T cell zones were disorganized
(Fig. 4, B and D) without detectable FDC clusters (Fig. 4,
F and H). This was associated with absence of anti-SRBC
IgG responses (see Fig. 3). Production of antigen-specific
IgM, however, was retained by all mice that received either
wild-type or LT
/
splenocytes, even in the context of
disturbed splenic microarchitecture (data not shown).
Fig. 4.
Structure of spleen
follicles in irradiated mice reconstituted with wild-type or LT/
splenocytes. After serum was
collected from mice shown in
Fig. 3, the spleens were harvested and frozen sections were
stained with anti-B220 (brown)
and anti-Thy1.2 (blue) to visualize the B cell and T cell zones
(A-D). Distinct B cell and T cell
zones were present in wild-type
mice that received splenocytes
from either normal (A) or LT
/
mice (C), whereas there was disturbed segregation of B cells and
T cells in LT
/
mice that received splenocytes from either
normal (B) or LT
/
mice (D).
FDC clusters were observed by
staining with the anti-CR1 monoclonal antibody 8C12
(blue) (E-H). FDC clusters were
retained in the spleen follicles of
wild-type mice that received
splenocytes from either wildtype (E) or LT
/
mice (G),
whereas FDC clusters were absent in the spleens of LT
/
mice that received splenocytes
from either wild-type (F) or
LT
/
mice (H).
[View Larger Version of this Image (101K GIF file)]
expressing cells are unable to reprogram discrete B cell and
T cell zones or clusters of FDC in irradiated LT
/
mice.
Similarly, established clusters of FDC are not in the short term dependent on the presence of LT
-expressing cells
(Fig. 4 G). The ability to introduce LT
-expressing cells into
a disturbed microenvironment and LT
-deficient cells into
a wild-type microenvironment allows us to examine the
role of intact follicle structure on antibody production. As
manifested by the presence of clustered FDC and/or normal B cell-T cell compartmentalization, wild-type follicle
structure determines the ability to develop a productive isotype switched antibody response. LT
-expressing lymphocytes are not per se required for switching.
-expressing Cells Reprogram the
Structure of the LT
/
Spleen to Support the Development of
an IgG Response.
in determination of spleen microarchitecture and antibody responsiveness permitting
long term reconstitution. 6 wk after lethally irradiated
LT
/
mice were given wild-type BM, they showed restoration of 8C12-staining FDC clusters (Fig. 5 B) and of
the ability to form morphologically intact germinal centers
(Fig. 5 F); however, the follicles remained small and segregation of B cell and T cell zones was incomplete (Fig. 6 B).
In contrast, when lethally irradiated wild-type mice were
reconstituted with LT
/
BM, follicle size was maintained
and segregation of B cell and T cell zones was at least partially retained (Fig. 6 C), but FDC clusters and GC formation were lost (Fig. 5, C and G). As expected, reconstitution of wild-type mice with wild-type BM resulted in the
re-establishment of morphologically normal follicles and
reconstitution of LT
/
mice with LT
/
BM yielded
small follicles with globally disturbed structure (Figs. 5 and
6). These data underscore the difference between shortterm and long-term reconstitution in this system. When
LT
/
mice are reconstituted with LT
-expressing splenocytes and examined 10 d later, they still have no FDC
clusters and are unable to generate GC. In contrast, when
LT
/
mice are reconstituted with LT
-expressing BM
cells and examined 6 wk later, FDC clusters have developed and GC can form in response to immunization. It is
currently unclear whether the differences we observed between BM and spleen cell transfers represent differences in
the functionality of BM and spleen cells (with BM containing a cell population capable of supporting development of
spleen FDC clusters and with splenocyte suspensions being
devoid of such cells) or more likely represents differences in
the time course of the experiment (with LT
-expression in
a transferred cell being required for more than 10 d in order for FDC clusters to form).
Fig. 5.
Spleen follicle structure in recipients of wild-type or
LT/
bone marrow. 6 wk after bone marrow reconstitution,
mice were immunized i.p. with
SRBC and 10 d later sections of
frozen spleen were stained with
8C12 (blue) to detect FDC, and
with anti-B220 (brown). Clusters
of FDC were detected in both
wild-type mice (A) and LT
/
mice (B) that had been reconstituted with wild-type BM. Clusters of FDC were not detected in
either wild-type (C) or LT
/
mice (D) that had received BM
from LT
/
mice. The GC reaction was assessed by staining
spleen sections with PNA (blue)
and anti-IgD (brown). GC were
observed in both wild-type
mice (E) and LT
/
mice (F)
that had received BM from wildtype donors, but were not detected in either wild-type (G) or
LT
/
mice (H) that were reconstituted with BM from
LT
/
donors.
[View Larger Version of this Image (111K GIF file)]
Fig. 6.
B cell/T cell organization in irradiated mice reconstituted with wild-type or LT/
bone marrow. Frozen sections of spleens from the recipients of BM transfer shown in Figure 5 were analyzed by staining with anti-Thy-1.2 (blue) and anti-B220 (brown). Wild-type mice that received BM from either wild-type (A) or LT
/
mice (B) showed segregation of B cells and T cells within lymphoid follicles, whereas the follicles of LT
/
mice
reconstituted with BM from either wild-type (C) or LT
/
mice (D) showed little or no segregation of B and T cell zones.
[View Larger Version of this Image (132K GIF file)]
and the TNF
genes, demonstrated recovery of the IgG response and almost complete recovery
of spleen B cell-T cell compartmentalization after these
mice were irradiated and reconstituted with wild-type bone marrow. It is currently unclear whether the apparent
differences in ability of LT
/
and TNF
/LT
doubledeficient mice to manifest restored B cell-T cell segregation after reconstitution with wild-type BM reflects a modulating effect of TNF
or represents subtle differences in tissue staining or BM transfer methods. Direct comparison
of the two mouse strains using identical techniques will be
required to resolve this question.
/
mice that received wild-type BM demonstrated an anti-SRBC IgG response similar in magnitude
to that seen in wild-type mice similarly reconstituted (Fig.
7). This was associated with restoration of GC containing
FDC clusters, but not with the formation of normal B cell
and T cell zones (see Figs. 5 and 6). In contrast, wild-type mice that received LT
/
BM had a severely impaired
IgG response (Fig. 7). This defective IgG response was in
the context of partially retained B cell-T cell compartmentalization but without detectable FDC clusters or GC (see
Figs. 5 and 6). Taken together, these findings suggest that
proper segregation of B cell and T cell zones is not required for productive isotype switching after i.v. immunization
with SRBC. Rather, they showed a strong correlation between the ability to form an anti-SRBC IgG response and
the presence of FDC clusters and GC determined by the
presence of LT
-expressing BM-derived cells. Thus, BMderived LT
-expressing cells are required in order for B
cells to switch to secretion of IgG, probably by virtue of
the action of this cytokine to support the development and maintenance of FDC clusters and GC. Additional cellular
and/or stromal elements besides FDC are no doubt required for the development of a functional GC reaction,
including for example elements of the dendritic cell network. We have no data, however, to support a specific role
for LT
as a regulator of development or function of these
additional elements.
Fig. 7.
IgG production after bone marrow transfer. Wild-type and
LT/
mice (3-4 mice/group) were lethally irradiated and reconstituted with wild-type or LT
/
as in Fig. 5. 6 wk later, they were immunized
i.p. with SRBC (108) and boosted 21 d later. Serum was collected 10 d
after both primary and secondary immunization and anti-SRBC IgG was
measured by ELISA as in Fig. 3. Results represent means ± SEM. Similar results were obtained in two additional experiments.
[View Larger Version of this Image (36K GIF file)]
/
splenocytes, although splenic
FDC clusters were easily detected 10 d after spleen cell
transfers, FDC clusters disappeared over the course of 2-3
wk. In contrast, 3 wk after transfer of wild-type splenocytes
to irradiated LT
/
mice, we began to detect clusters of
FDC, and concomitant ability to form the development of
an antigen-specific IgG response (data not shown).
/
splenocytes to
mildly irradiated (300 rad) or non-irradiated RAG-1-deficient mice. This allows observation of the action of the
transferred lymphoid cells in the absence of any host-derived lymphocytes. These experiments gave similar results compared to those described above. LT
-expressing splenocytes elicit the formation over the course of approximately
3 wk of clusters of FDC and the potential to form GC and
antigen-specific IgG (data not shown).
/
mice immunized with high doses of
SRBC is defective. This contrasts with our previous report
in which immunization of LT
/
mice i.p. with high
doses of NP-OVA adsorbed to alum led to the development of a strong IgG response to NP (8). Recent studies using TNF
/
mice (14) have shown similar variability of
the IgG response depending on the details of the immunization program. When TNF
/
mice were immunized
with SRBC in PBS, they showed a severely impaired IgG
response. When they were immunized with TNF-KLH in complete Freund's adjuvant, they showed productive isotype switching. We have performed preliminary experiments to investigate this phenomenon and to determine if
adjuvant restores the anti-SRBC IgG response in LT
/
mice. These studies showed that when LT
/
mice were
immunized i.p. with 108 SRBC emulsified with IFA, a brisk
IgG response developed similar to that of wild-type mice.
/
mice,
however, was associated with neither the de novo formation of FDC clusters nor the development of GC (data not
shown). This suggests that antigens administered i.p. with
adjuvant can circumvent the need for proper follicle structure in the generation of a mature IgG response. This could
be the consequence of the ability of adjuvant to induce local production of cytokines that substitute for signals produced in an intact follicle, or perhaps the ability of adjuvant
to affect antigen presentation or interaction of antigen with
responding B cells or T cells. In this regard, it is of interest that Banks et al. (12) observed that LT
/
mice immunized twice s.c. with KLH plus incomplete Freund's adjuvant generated a weak IgG response. The failure of adjuvant to restore the IgG response in this setting might have
been due to disturbed antigen trafficking without regional
LN. In addition, the defect in production of IgG in mice
deficient in either CD40 or CD40L was also not overcome
by immunization with adjuvant (18, 19). This suggests that
the mechanisms by which LT
and CD40/CD40L support
Ig isotype switching may be different.
contributes importantly to the development of
normal spleen microarchitecture. LT
also provides signals
that support the maturation of primary and secondary splenic
follicles after antigen challenge. Our data suggest that the
ability of LT
to support the development of an intact IgG
response is permissive, a consequence of this cytokine's action to establish elements of intact spleen follicle structure.
Address correspondence to David D. Chaplin, Washington University School of Medicine, 660 Euclid Ave., Box 8022, St. Louis, MO 63110.
Received for publication 22 January 1997 and in revised form 9 April 1997.
1 Abbreviations used in this paper: AP, alkaline phosphatase; BM, bone marrow; FDC, follicular dendritic cells; LN, lymph nodes; LTWe thank Jori Scripter, David Randolph, and Shuhua Han for helpful discussions.
This work was supported in part by a National Institutes of Health grant (D.D. Chaplin). D.D. Chaplin is an investigator of the Howard Hughes Medical Institute.
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