By
§
From the * Department of Laboratory Medicine/Pathology, the Department of Internal Medicine, and
the § Howard Hughes Medical Institute and Center for Immunology, Washington University School of
Medicine, St. Louis, Missouri 63110
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Abstract |
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Lymphotoxin (LT) is expressed by activated T cells, especially CD4+ T helper type 1 cells,
and by activated B and natural killer cells, but the functions of this molecule in vivo are incompletely defined. We have previously shown that follicular dendritic cell (FDC) clusters and germinal centers (GCs) are absent from the peripheral lymphoid tissues of LT
-deficient (LT
/
)
mice. LT
/
mice produce high levels of antigen-specific immunoglobulin (Ig)M, but very
low levels of IgG after immunization with sheep red blood cells. We show here that LT
-expressing B cells are essential for the recovery of primary, secondary, and memory humoral
immune responses in LT
/
mice. It is not necessary for T cells to express LT
to support these immune functions. Importantly, LT
-expressing B cells alone are essential and sufficient
for the formation of FDC clusters. Once these clusters are formed by LT
-expressing B cells,
then LT
-deficient T cells can interact with B cells to generate GCs and productive class-switched antibody responses. Thus, B cells themselves provide an essential signal that induces
and maintains the lymphoid microenvironment essential for GC formation and class-switched Ig responses.
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Introduction |
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Lymphotoxin (LT)1 exists in two molecular forms, a
soluble homotrimer that is structurally related to soluble TNF and that can bind and activate the two recognized
TNF receptors TNFR-I and TNFR-II (1, 2), and a membrane-bound heterotrimer in association with the structurally related LT
chain with stoichiometry LT
1LT
2 (3,
4). This ligand interacts with the TNFR-related protein
(TNFR-rp, also known as the LT
receptor; references 4, 5), a receptor that is expressed widely on nonlymphoid cells. Several of the ligands and receptors of the TNF/LT family
contribute to the formation of normal lymphoid tissue
structure. For example, LT
(6), LT
(9), and TNFR-I
(10) are each required for the formation of normal Peyer's
patch structure, and all of these plus TNF (11) are required
for the formation of clusters of follicular dendritic cells
(FDC) within the primary and secondary follicles of the
spleen white pulp. In mice deficient in these cytokines or
receptors, there was failure to form mature isotype-switched Ig responses after immunization with T cell-dependent antigens (such as sheep RBCs [SRBCs] or keyhole limpet
hemocyanin) administered without adjuvants (7, 11).
These results, then, suggest that both TNF- and LT-expressing cells may interact with FDCs or FDC precursors to
support their development into organized clusters within
primary spleen follicles. Bone marrow (BM)-derived LT
-expressing cells are able to restore the formation of clusters
of FDCs after they are transferred into LT
-deficient
(LT
/
) mice (13). These restored FDC clusters can then
support the formation of germinal centers (GCs) and mature isotype-switched Ig responses when the reconstituted
mice are immunized with SRBCs and other antigens.
Thus, LT
produced by BM-derived cells is essential for
the formation of clusters of FDCs, which in turn contributes a permissive environment for the development of mature B cell responses.
Our initial experiments indicated that when wild-type
BM cells were transferred into LT/
recipients, FDC clusters formed in the spleen white pulp between 2 and 4 wk
after cell transfer, and effective IgG responses could be detected after that. In contrast, when reconstitution of LT
/
mice was with mature wild-type spleen cells, no detectable
FDC clusters or IgG to SRBCs could be identified 10 d after cell transfer and immunization with SRBCs (13). This
suggested that several weeks are required for LT
-expressing BM-derived cells to restore the microenvironment, including the formation of FDC clusters, for effective IgG responses. With this in mind, it is likely that short-term
reconstitution experiments in which LT
/
mice receive
either mixed splenocytes or purified lymphocyte subsets
will not be suitable for analysis of the cellular elements that
control the formation of functional FDC clusters.
It is known that both T and B cells are required for the
generation of GCs and for the production of class-switched
antibodies in response to T-dependent antigens. Prior studies of nude mice using partially purified preparations of mature T cells have shown that only a small fraction of T cells
are required to cooperate with B cells in the formation of
GCs and an isotype-switched IgG response (14, 15). To
eliminate problems with contamination of one lymphocyte
lineage with another, we elected to define the requirement
for LT-expressing cell lineages by transfer of BM from
donor animals with genetic ablation of individual cell lineages rather than exclusively by transfer of purified populations of splenic LT
-expressing lymphoid cell subsets. We
show here that LT
-expressing B cells are essential for the
recovery of primary, secondary, and memory humoral immune responses in LT
/
mice. Once proper FDC clusters are formed by B cells expressing LT
, then LT
-deficient T cells can interact with B cells to generate productive
GCs and class-switched antibody responses.
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Materials and Methods |
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Mice.
C57BL/6J, 129Sv, recombination activating gene (RAG)- 1Measurement of antigen-specific Ig.
Specific antibodies were measured and analyzed as previously described (16). In brief, Immulon 4 plates (Dynatech Laboratories, Inc., Chantilly, VA) were coated with SRBCs (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 Assoc., 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 Chemical 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 using linear regression analysis. The results represent mean ± SEM.Transfer of Lymphocytes.
Whole spleen cell suspensions were prepared from single mouse donor by mincing with scissors and teasing the spleen between two frosted microscope slides. T and B cells were enriched using a nylon wool column as described (17). B cells were further purified by treatment with anti-Thy1.2 plus complement as previously described (18). These preparations contained 92-95% B cells, defined as IgM+B220+ by flow cytometry. There were no detectable contaminating T cells. For T cell reconstitution, preparations were >80% T cells, and contained <10% B cells. The contaminating B cells are unlikely to be significant because the recipient TCRBM Transplantation.
Bone marrow was harvested and recipients were prepared as described previously (13, 18). Recipient mice were lethally irradiated with 1,050 rads and reconstituted with 5 × 106 donor BM cells. 6 wk after transplantation, recipients were immunized intraperitoneally with 108 SRBCs, and serum samples were collected 10 d after primary or secondary immunization.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 and biotinylated Thy1.2 (PharMingen, San Diego, CA), biotinylated anti-complement receptor (anti-CR)1 (8C12; PharMingen), or biotinylated peanut agglutinin (PNA; Vector, Burlingame, CA), all at 1:50-1:100 dilutions. Horseradish peroxidase-conjugated rabbit anti-FITC (diluted 1:20; Dako, Glostrup, Denmark) and alkaline phosphatase-conjugated streptavidin (diluted 1:20; Zymed, South San Francisco, CA) was added 1 h later. Color development for bound alkaline phosphatase and horseradish peroxidase was with an alkaline phosphatase reaction kit (Vector) and with diaminobenzidine. ![]() |
Results and Discussion |
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LT is expressed by activated T cells, especially CD4+ T
helper type 1 cells, and by activated B and NK cells (2). Both T and B cells are required to induce formation of GCs and
class-switched antibody responses to T cell-dependent antigens. The current experiments were designed to determine
which BM-derived cellular elements, namely T, B, or NK
cells, are required to deliver the LT
-dependent signal(s) for
both formation of GCs and for production of a mature, isotype-switched Ig response. We used BM from wild-type
mice as a source of LT
-expressing cells of all marrow-
derived lineages. BM from Ig µ heavy chain-targeted mice
(BCR
/
) provided all marrow-derived LT
-expressing lineages except B cells, and bone marrow from mice deficient
in both TCR-
and TCR-
(TCR
/
) provided all marrow-derived LT
-expressing lineages except T cells. BM
from RAG-1
/
mice provided all marrow-derived LT
-expressing lineages except B and T cells. Compound BM
chimeric animals were prepared by reconstituting lethally irradiated LT
/
mice with a 1:1 mixture of BM from
LT
/
mice (to provide LT
/
cells from all hematopoietic lineages) and wild-type, BCR
/
, TCR
/
, or
RAG-1
/
mice. These reconstituted animals contained all
hematopoietic lineages, but one or more of these lineages
was incapable of expressing LT
.
Mice reconstituted with a mixture of LT/
and wild-type BM were able to generate a strong IgG anti-SRBC
response when immunized intraperitoneally 6 wk after
marrow transplant (Fig. 1 A). Thus, wild-type cells were
dominant over LT
/
cells for formation of an isotype-switched T cell-dependent antibody response. Similarly,
mice reconstituted with a mixture of LT
/
and TCR
/
BM were also able to generate a strong IgG anti-SRBC response. This indicates that LT
-expressing T cells are not
required for the development of an isotype-switched antibody response. However, mice reconstituted with a mixture of LT
/
and either BCR
/
or RAG-1
/
BM
failed to generate an antigen-specific IgG response. Thus, LT
-expressing B lymphocytes are required for the recovery of an isotype-switched antibody response.
|
LT/
mice manifest impaired secondary (Fig. 1 B) and
memory (Fig. 1 C) responses after challenge with SRBCs.
To investigate whether LT
-expressing B cells were able
to reconstitute these responses, the compound BM chimeric animals were evaluated further. Strong secondary
and memory IgG responses were detected in mice reconstituted with a mixture of BM from LT
/
and TCR
/
mice (Fig. 1, B and C). As expected, secondary and memory IgG responses were absent in mice that had been reconstituted with mixtures of LT
/
and either BCR
/
or RAG-1
/
marrow (data not shown). These data indicate both that LT
-expressing B cells support the development of secondary and memory responses, and also that T
cells do not require LT
expression to provide help for the
responses. Although signals from T cells by membrane proteins such as CD40 ligand (CD40L) are essential for generating memory B cells, LT
from T cells is not essential to
provide B cell help for these responses. Consistent with
these data, we found in other experiments that LT
/
mice could not generate memory B cells, but could generate memory T cells (data not shown).
Clusters of
FDCs are prominent components of lymphoid follicles that
are thought to support the formation of GCs that are required for the development of robust primary and secondary IgG responses in an essential fashion (19, 20). We have
recently shown that the reconstitution of antigen-specific
IgG responses in LT/
mice by transplantation of wild-type BM is closely associated with the recovery of FDC
clusters (13). These prior studies demonstrated that BM-derived cells produced a population of LT
-expressing cells that was capable of supporting the development of
clusters of FDCs within the splenic white pulp nodules. To
define the essential LT
-expressing lineage responsible for
delivering this signal, we analyzed the structures of spleen
follicles from LT
/
mice that had been lethally irradiated
and reconstituted with a mixture of BM from LT
/
mice and either BCR
/
or TCR
/
mice. FDC clusters
were detected by immunohistochemistry using the anti-CR1 monoclonal antibody 8C12 (21), and GCs were detected by binding PNA (Fig. 2). Both FDC clusters and
GCs were restored in LT
/
mice that had received a
mixture of BM cells from LT
/
mice and TCR
/
mice. This demonstrated that it was not necessary for cells
of the T lymphocyte lineage to express LT
for FDC clusters and GCs to form. In contrast, neither FDC clusters nor
GCs were detected in mice that had received a mixture of
BM from LT
/
and BCR
/
mice. Thus, LT
-expressing B cells are required for formation of splenic primary
follicles including clusters of FDCs. Although both T and
B cells are required for GC formation and for production of IgG in response to T cell-dependent antigens like
SRBCs, the signals provided by each of these cell populations have been incompletely defined. Our data demonstrate that interactions between LT
-expressing B cells from
TCR
/
mice and LT
-deficient T cells from LT
/
mice are capable of supporting GC formation, isotype class
switching, and B cell memory in the reconstituted environment. Of particular interest, our data demonstrate that B
cells in an important way use LT
to condition their lymphoid tissue microenvironment in a way that supports the
development of effective IgG responses.
|
As observed in LT/
mice, the peripheral lymphoid tissues of scid mice are devoid of FDC clusters (21), suggesting that mature B and/or T lymphocytes
are required for FDC cluster formation to occur. Using scid
recipients, Kapasi et al. suggested that either T or B cells
could partially reconstitute FDC cluster formation in lymph
nodes, but that both cell populations were required for full
restoration of FDCs (21). A special role for B cells inducing development of FDCs had been previously postulated by
Cerny et al. (22). Additionally, Yoshida et al. showed, using an allogeneic system, that when graft versus host disease
was suppressed by treatment of recipients with anti-T cell
antibodies, that splenocytes (presumably with T cell function blocked) could restore endogenous FDC cluster formation in scid recipients (23). However, the ability of B or
T cells alone to induce FDC clusters is difficult to assess because of the difficulties in defining pure cell populations in
these studies. In addition, T lymphocytes (even in very
small numbers) have been unequivocally demonstrated to
be essential for the formation of antigen-driven GCs (14,
15). Experimental interference with T cell function either
by selective T cell depletion or by blocking costimulatory
signals such as those delivered by CD40-CD40L or B7-
CD28 prevents the formation of GCs and the development of specific IgG responses (20, 24). It is of interest,
therefore, that FDCs express CD40 (30), which has the potential to interact in a productive fashion with CD40L on
T cells, and also that patients with a congenital deficiency
of CD40L have lymph nodes with severe depletion of
FDC clusters (25). These observations suggest that some
CD40-CD40L interaction may be essential for the development of FDC clusters. It was, therefore, unexpected that
we found that mice with targeted deficiency of CD40L
(31), although they showed no formation of GC after intraperitoneal immunization with SRBCs, nevertheless
manifested splenic primary follicles with clusters of FDC
similar to those seen in normal mice (data not shown). This
suggests that the development and/or maintenance of FDC
clusters and segregated T and B cell zones may be independent of a CD40-CD40L interaction.
Because FDC clusters were preserved in CD40L/
mice
that have profound impairment of B-T cell interaction, we
investigated whether, in fact, any B-T cell interaction was
required for the formation of splenic FDC clusters. Sections from the spleens of TCR
/
and BCR
/
mice were
stained with the anti-CR1 monoclonal antibody 8C12 to
detect the presence of FDC clusters (Fig. 3). Strikingly, the spleens of TCR
/
mice manifest FDC clusters similar to
those seen in wild-type mice. Spleen sections from BCR
/
mice showed no detectable clusters of FDCs. These data
indicated both that LT
-expressing B cells are required for
the formation of FDC clusters and that these B cells can
support the formation of FDC clusters in the absence of T
cells. That B cells are the only lymphoid cell type necessary
for the induction of FDC clusters is suggested by our preliminary analysis of CD3
transgenic mice. These mice lack
NK and mature T cell activities (32, 33). Interestingly, immunohistochemical analysis of sections from their spleens
shows clusters of FDCs within B cell zones, similar to those
seen in the TCR
/
mice (data not shown), suggesting that
neither NK or T cells are required for the development of
FDC clusters.
|
As expected,
when TCR/
mice were immunized with SRBCs, although there was a substantial IgM anti-SRBC response,
there was no development of morphologically defined GCs
and no detectable production of IgG anti-SRBC antibody
(data not shown). In contrast, when BM from TCR
/
was adoptively transferred into LT
/
mice, there was full
recovery of GC formation and ability to produce antigen-specific IgG (Figs. 1 and 2). These data indicated that LT
/
T cells from the recipient animal could act together with
the LT
-expressing B cells from the donor to form GCs
and to support Ig class switching. Additional evidence that
T cells can support the formation of GCs in an LT
-independent fashion was obtained by transfer of purified T cells
from either wild-type or LT
/
mice into FDC clusters
containing TCR
/
recipients. Unirradiated TCR
/
mice were treated by infusion of 107 purified splenic T cells
from either wild-type or LT
/
donors together with an
infusion of 108 SRBCs. 10 d later, both GCs (Fig. 4) and
high levels of IgG anti-SRBC antibodies (data not shown)
were detected in the TCR
/
recipients, irrespective of
whether they received wild-type or LT
/
T cells. This
indicates that the FDC clusters that are found in TCR
/
mice are competent to support the formation of GCs and a
productive isotype-switched Ig response once supplemental
T cells are provided. In contrast to T cells from CD40L
/
mice, LT
-deficient T cells can activate B cells for the GC
reaction and can support isotype switching once the proper
microenvironment has been established by LT
-expressing
B cells.
|
The BM transfer studies described above indicate that LT-expressing cells of the B cell lineage are required to induce formation of spleen FDC clusters. To investigate whether the mature B cell component of this
lineage is sufficient to provide the LT
-dependent signal
for FDC formation, we reconstituted unirradiated RAG-1
/
mice with an intravenous infusion of either 107 purified splenic B cells or 2.5 × 106 BM cells from TCR
/
donors. 3 wk after transfer, both groups of mice show similar splenic FDC clusters (Fig. 5). These data indicate that
LT
-expressing mature B cells alone are sufficient to reconstitute formation of endogenous FDC clusters in the
primary follicles. Interestingly, resting virgin B cells are
thought to be negative for expression of LT
(29, 34). This
implies, then, that the B cell must participate in a dialogue
in which it is induced, presumably in the proper area of the
white pulp, to express LT
, which then can deliver the signal for formation of FDC clusters. The nature of the signal
that induces LT
expression, and the immediate target cell
receiving the LT
signal, remain to be defined. The LT-dependent signal appears to be required not only for the induction, but also for the maintenance of FDC clusters. This
is suggested by our prior data showing that FDC clusters
disappear over the course of 2-3 wk after replacement of
normal BM by LT
/
BM (13). This, then, is a unique
example of B cells providing a signal that serves to induce
and maintain structural elements that support the further
maturation of B cell functional responses.
|
Of particular interest in relation to the ability of B cells
to induce FDC clusters within the lymphoid tissue compartment of the spleen are the prior descriptions of ectopic
development of FDC-containing lymphoid follicles and
GCs in certain chronic inflammatory conditions and autoimmune diseases. For example, patients with myasthenia
gravis can manifest lymphoid follicles and GCs within the
thymus (35). Similar follicles and GCs are found within the
inflamed synovial tissues of patients with rheumatoid arthritis (36). These GCs contain clusters of FDCs similar to those found in normal lymphoid tissue GCs. We speculate
that expression of LT by B cells infiltrating these tissues
may provide the initial signals for formation of FDC clusters that support the formation of these ectopic GCs. FDC
clusters, however, do not colocalize with B cells throughout the entire B cell zones of peripheral lymphoid tissues.
Rather, they form clusters near the centers of the primary
B cell follicles and spare the periphery of these follicles.
One possible explanation for this positioning is that local
signals may regulate the expression of LT
within these B
cell areas. Alternatively, this restricted positioning may imply that there are additional signals besides B cell-derived
LT
that are required to induce the development and
maintenance of FDC clusters. More complete understanding of the signals that regulate the formation and maintenance of FDC clusters may provide new opportunities for
manipulation of the quality and magnitude of immune responses.
![]() |
Footnotes |
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Address correspondence to David D. Chaplin, Howard Hughes Medical Institute and Center for Immunology, Department of Internal Medicine, Washington University School of Medicine, St. Louis, MO 63110. Phone: 314-362-9047; Fax: 314-454-0486; E-mail: chaplin{at}im.wustl.edu
Received for publication 20 October 1997 and in revised form 12 December 1997.
1 Abbreviations used in this paper: anti-CR, anti-complement receptor; BCR, B cell receptor; BM, bone marrow; CD40L, CD40 ligand; FDC, follicular dendritic cell; GC, germinal center; LT, lymphotoxin; PNA, peanut agglutinin; RAG, recombination activity gene; SRBC, sheep RBC.The authors thank David Randolph (Washington University School of Medicine, St. Louis, MO), Hector Molina (Washington University School of Medicine, St. Louis, MO), and Mitsuru Matsumoto (Ehime University School of Medicine, Ehime, Japan) for helpful discussions.
This work was supported by grant AI01431 (Y.-X. Fu) from the National Institutes of Health. D. Chaplin is an investigator of the Howard Hughes Medical Institute.
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