| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
-Dependent Spleen Microenvironment Supports the Generation of Memory B Cells and Is Required for Their Subsequent Antigen-Induced Activation1
,
Departments of
*
Laboratory Medicine/Pathology and
Internal Medicine, and
Howard Hughes Medical Institute and Center for Immunology, Washington University School of Medicine, St. Louis, MO 63110
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
-deficient (LT-/-) mice show
dramatically reduced IgG responses after either primary or secondary
immunizations with sheep red blood cells (SRBC). When splenocytes from
SRBC-primed wild-type donor mice were infused into irradiated naive
wild-type recipient mice, they generated a robust memory IgG response,
but not when infused into LT-/- recipients, indicating
that the microenvironment that develops in LT-/- mice
is incompetent to support the activation of this memory response. When
irradiated wild-type mice were reconstituted with splenocytes from
primed LT-/- donors and then challenged with the same
immunizing Ag, no memory response was observed, indicating further that
memory cells could not be generated in the LT-/-
environment. To address which lymphocyte subsets were impaired in the
LT-/- mice, we performed reconstitution experiments
using a hapten/carrier system and T cells and B cells from different
primed donors. There was no detectable defect in either the generation
or expression of memory T cells from LT-/- donors. In
contrast, B cells were not primed for memory in the microenvironment of
LT-/- mice. Additionally, primed wild-type memory B
cells could not express a memory IgG response in the
LT-/- microenvironment. Thus, splenic white pulp
structure, which depends on the expression of LT for its development
and maintenance, is needed to support the generation of memory B cells
and to permit existing memory B cells to express an isotype switched
memory Ig response following antigenic challenge. |
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
- and
LTß-chains with predominant stoichiometry
LT1ß2 (5). Signaling by the
LT1ß2 heterotrimer is mediated through the
LTß receptor (LTßR). Thus, membrane LT signals are independent of
the type I or type II TNF receptors that mediate signals from the
soluble LT3 homotrimer (5, 6). Mice
rendered deficient in LT, LTß, or the LTßR are born with a
dramatic impairment of lymph node and Peyer’s patch biogenesis. In
addition, LT-/-, LTß-/-, and
LTßR-/- mice each fail to form distinct splenic T cell
and B cell zones, follicular dendritic cell (FDC) clusters, or germinal
centers (GC). These structural disturbances are associated with
impaired high affinity isotype-switched Ig responses following primary
or secondary immunization with T cell-dependent Ags (such as sheep red
blood cells (SRBC) or keyhole limpet hemocyanin (KLH)) when
administered without adjuvants (7, 8, 9, 10).
Long-term reconstitution of lethally irradiated LT-/-
mice with bone marrow cells from wild-type (wt) mice leads to
restoration of the ability to form FDC clusters, GC, and
adjuvant-independent strong IgG responses (8). We have
recently shown that B cells alone are required to deliver the
LT-dependent signals that restore the formation of FDC clusters, GC,
and recovery of IgG responses in LT-/- mice
(11). Expression of LT by T cells is not required. In
contrast, when sublethally irradiated LT-/- mice are
reconstituted with spleen cells from wt donors rather than with bone
marrow cells, no FDC clusters, GC, or T cell-dependent high affinity
IgG responses are detected 2 wk after cell transfer and immunization
with SRBC (8). These findings suggest that the
microenvironment that develops in LT-/- mice cannot
support a high affinity isotype-switched Ig response, but that at least
some aspects of this microenvironment are plastic and can be restored
under the influence of sustained LT-dependent signaling through the
LTßR. The ability to mount a high affinity isotype-switched Ig
response appears to be correlated with the presence of clusters of FDC,
which can be detected between 2 and 3 wk following transfer of
LT-expressing B cells into LT-/- recipients. The
present study was undertaken to determine whether the lymphoid tissue
microenvironment that forms under the influence of LT is required to
support either the induction or expression of memory responses.
Several studies suggest that memory B cells form primarily in GC and that their formation requires the presence of FDC clusters (12, 13, 14, 15). A prominent contribution of GC to the formation of memory B cells is suggested by the observation that the B cell Ag receptors expressed by memory B cells carry a significant number of somatic mutations and generally show high affinity for the eliciting Ag. Thus, memory B cells appear to have passed through GC during their development. Like maturing B cells, Ag-specific T cells also undergo phenotypic changes within GC (16, 17, 18); however, it remains unclear whether these GC-dependent changes in T cells also represent an integral part of the program leading to the generation of memory T cells. It also remains unclear whether GC or FDC clusters play an important role in the expression of memory responses by established memory T and B cells.
Until recently, the lack of animal models in which the clustering of
FDC and the formation of GC could be regulated has limited our
understanding of the role of these structures in the generation of
memory cells and in the maintenance of memory responses.
LT-/- mice fail to form clusters of FDC and are unable
to generate GC following immunization with T-dependent Ags
(7). These mice provide a model in which to study whether
these structures contribute to the generation of memory T or B cells
and to the functional expression of the memory response. We demonstrate
here that the microenvironment of LT-/- mice supports
the generation of memory T cells but not of memory B cells.
Furthermore, the microenvironment in LT-/- mice cannot
support the expression of functional memory IgG responses, even when
sensitized memory cells from wt mice are provided by adoptive transfer.
Of interest, when sublethally irradiated LT-/- mice
are reconstituted with splenocytes from wt mice, immunization elicits
the formation of clusters of peanut agglutinin+
(PNA+) cells, apparently in the absence of associated FDC,
suggesting that normal B cells can be activated to form GC-like
structures without FDC; however, these clusters of PNA+
cells fail to differentiate into functional memory cells in the splenic
microenvironment of LT-/- mice. The dysfunctional
nature of these GC-like structures is underscored by their dissociation
from the apoptotic process that normally appears to be a consequence of
B cell selection in these structures (15, 19).
|
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
C57BL/6J, 129Sv, and B cell receptor (BCR)-/-
(C57BL/6J-Igh-6tmlCgn) mice were obtained from
The Jackson Laboratory (Bar Harbor, ME). LT-/- mice
(1) were maintained on a mixed 129Sv x C57BL/6
background and were bred under specific pathogen-free conditions.
Preparation of NP-haptenated SRBC
Four milliliters of 10% SRBC (Colorado Serum, Denver, CO) in PBS were incubated with 4 ml of NP-Osu (1 mg/ml, Biosearch Technologies, San Rafael, CA) in 0.15 M NaHCO3 for 2 h at room temperature. The NP-conjugated SRBC were washed with PBS and resuspended in 8 ml of PBS. Immunization was with 0.1 ml of this suspension injected i.p. NP13-KLH was also purchased from Biosearch Technologies.
Measurement of Ag-specific Ig
Specific Abs were measured and analyzed as previously described (8). For measurement of anti-SRBC Abs, 96-well Falcon plates (Becton Dickinson, Lincoln Park, NJ) were coated with SRBC (150 µl of 0.1% SRBC in PBS per well). For anti-NP Abs, 96-well Immulon 4 plates (Dynatech Laboratories, Chantilly, VA) were coated with NP2.5-BSA (10 µg/ml, Biosearch Technologies) for 1 h. Unbound Ags were removed by washing with PBS. Diluted mouse sera were then added and incubated at 4°C for 1 h. Bound Abs were detected using 100 µl of 1:2000 diluted alkaline phosphatase-conjugated goat anti-mouse IgG-specific antiserum (Southern Biotechnology Associates, Birmingham, AL), followed by addition of the alkaline phosphatase substrate p-nitrophenyl phosphate (Sigma, St. Louis, MO) at 1 mg/ml. The mean OD405 from triplicate wells was compared to a various dilutions of a standard anti-NP immune serum to calculate the relative units (RU) using linear regression analysis. The results are reported as means ± SEM.
Transfer of splenocytes
Whole spleen cell suspensions were prepared from single donor mice and were injected i.v. into recipients that had been irradiated with 750 rads 3 h earlier. When indicated, SRBC were injected i.v. together with the spleen cell suspensions. Each recipient received all of the cells derived from a single donor spleen.
Enrichment for T and B cells
Nylon wool columns (Polysciences, Warrington, PA) were used to enrich spleen cell suspensions for T or B cells. Splenocytes were incubated on nylon wool columns with 10% FCS in DMEM for 1 h at 37°C. Nonadherent cells were eluted with 10% FCS in DMEM at 37°C. Enriched T cells in the fraction of nonadherent cells were further purified by panning on tissue culture dishes coated with goat anti-mouse Ig H/L chain (Southern Biotechnology Associates), yielding 75–85% T cells. The contamination by B cells was <5%. Cells that adhered to nylon wool were eluted using cold PBS. Eluted cells were 80–90% B cells. After panning on tissue culture dishes coated with anti-Thy1.2 Ab, contamination by T cells was reduced to <5%.
Evaluation of spleen follicle structure and apoptosis
Spleens were harvested, embedded in OCT compound (Miles, Elkhart, IN), and frozen in liquid nitrogen. Frozen sections (6–10 µm thick) were fixed, quenched, and stained as previously described (8) using 0.2% H2O2 in methanol. After washing, the sections were stained by first incubating with FITC-conjugated B220 (PharMingen, San Diego, CA), and biotinylated PNA (Vector, Burlingame, CA), all at 1:100 dilution. 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.
Cells undergoing apoptosis were detected using a modified TUNEL method (20). Tissue sections were incubated with 2 mM digoxigenin-conjugated dUTP (Boehringer Mannheim, Indianapolis, IN) and 5 U of TdT in 0.5 M cacodylate (pH 6.8), 1 mM CoCl2, 0.5 mM DTT, 0.05% BSA, and 0.15 M NaCl. After washing in Tris-buffered saline, sections were incubated with sheep anti-digoxigenin Ab (Boehringer Mannheim) in Tris-buffered saline, washed, and further incubated with HRP-conjugated anti-sheep Ig Ab (Jackson ImmunoResearch, West Grove, PA). Color development for bound HRP was with 100 µg/ml 3-amino-9-ethylcarbazole in 0.17 M sodium acetate (Sigma). Sections were then counterstained with 1% methyl green.
|
Results and Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
-/- mice
We previously reported that LT-/- mice developed
a dramatically reduced Ag-specific IgG response following immunization
with the T cell-dependent Ags SRBC or KLH without adjuvant
(8). We now extend these experiments to test the character
of the IgG response in LT-/- mice that received
repeated immunizations intended to elicit immunological memory. Three
months after initial priming, LT-/- and wt mice were
challenged by i.p. immunization with 108 SRBC without
adjuvant. Anti-SRBC IgG was measured 5 days after the booster
immunization (Fig. 1
A).
Measurements were made 5 days after challenge because at this time
memory responses in wt mice show strong IgG production, whereas primary
IgG responses remain undetectable or low. Wt mice mounted an
anti-SRBC memory response that was more than 200-fold higher than
that of LT-/- mice, suggesting that there was a severe
impairment of the memory response in the LT-/- mice.
We had previously shown that LT-/- mice were not
absolutely unable to generate an IgG response, but could do so
following immunization with either NP-OVA (21) or SRBC
(8) when the Ag was administered with an adjuvant.
Therefore, we tested the memory IgG response following priming with IFA
(Fig. 1B). Although LT-/- mice showed a
robust IgG response when immunized with SRBC in IFA, when challenged 3
mo later, they showed only a weak anti-SRBC memory response. These
experiments suggest that LT-/- mice either are
unable to express a memory response or fail to generate long-lived
memory lymphocytes.
|
-/- mice, they fail to
mount a memory response
To test whether the lymphoid tissue microenvironment that forms in
congenitally LT-deficient mice can support the function of memory
lymphocytes, we transferred splenic lymphocytes from either naive or
SRBC-immunized wt mice into irradiated naive wt or
LT-/- recipients and then challenged these chimeric
recipients with SRBC. In previously immunized wt animals, a strong
memory IgG response can typically be detected as early as day 3–5
after i.p. challenge. When naive cells were transferred, development of
a significant primary IgG response was not detected 5 days after
immunization of the recipients, although it was by 10 days (Fig. 2). In contrast, when primed lymphocytes
from SRBC-immunized wt mice (expected to contain memory B and T cells)
were transferred into naive wt recipients, challenge with SRBC elicited
a high titer IgG response on day 5 with a further increase by day 10.
Strikingly, when lymphocytes were transferred from primed wt mice to
LT-/- recipients, rechallenge with SRBC failed to
produce a high titer memory IgG response (even by day 10 after
rechallenge). Thus, preformed memory cells elicited in wt mice were
unable to express a mature memory response in the disturbed lymphoid
tissue environment that exists in LT-/- mice. The
disturbed microenvironment in LT-/- mice can support
neither primary (8) nor memory responses, even when
LT-expressing naive or memory cells are provided.
|
-/- mice
The studies described above showed that primed wt memory cells
were unable to express a memory IgG response following transfer into an
LT-/- recipient mouse. To compare the ability of the
lymphoid microenvironments of wt and LT-/- mice to
support the formation of memory lymphocytes, we immunized wt and
LT-/- mice with the T-dependent Ag SRBC to provide a
stimulus for memory cell formation. Splenocytes from the primed mice
were then transferred to sublethally irradiated wt recipients. These
reconstituted animals were challenged with SRBC to stimulate memory
cells that might have been transferred (Fig. 3). Five days after challenge, mice that
had received cells from primed wt donors showed a brisk IgG
anti-SRBC response characteristic of established B cell memory. In
contrast, cells transferred from primed LT-/- mice
supported no IgG response, similar to cells transferred from naive
LT-/- donors. This clearly indicates that functional
memory cells had not been induced in the LT-/-
environment.
|
-/- mice
Memory cells of both the B and T cell lineages are required to
coordinate memory responses in vivo (14, 22). To address
which memory cell compartment(s) failed to form in the microenvironment
of LT-/- mice, a hapten-carrier system was applied. To
investigate whether LT-/- mice could generate memory B
cells, the following strategy was used. BCR-/- mice were
primed i.p. with SRBC in PBS to generate anti-SRBC memory T cells
that were free of contamination by naive or memory B cells. Wt mice and
LT-/- mice were primed i.p. with NP-KLH in PBS to
provide potential sources of anti-NP memory B cells. Then,
107 SRBC-primed T cells from the spleens of
BCR-/- mice were mixed with an equal number of partially
purified splenic B cells from NP-KLH-immunized wt or
LT-/- mice and transferred to sublethally irradiated
(750 rads) naive wt recipients. Following challenge with NP-SRBC, mice
that received B cells from the NP-KLH-immunized wt mice showed a robust
memory anti-NP IgG response, whereas mice that received B cells
from immunized LT-/- mice showed at least a 50-fold
lower anti-NP IgG response (Fig. 4).
These data demonstrate a substantial impairment in the formation of
memory B cells in LT-/- mice.
|
-/- mice could generate memory T
cells, wt mice and LT-/- mice were primed i.p. with
SRBC in PBS to provide a source of potential anti-SRBC memory T
cells. Wt and LT-/- mice were primed i.p. with NP-KLH
in PBS to provide a source of potential anti-NP memory B cells.
Primed T and B cells (107 each) purified from the
SRBC-immunized and the NP-KLH-immunized mice were then transferred to
irradiated wt recipients. Five days after i.p. challenge with NP-SRBC
in PBS, a robust IgG anti-NP response was detected in mice that
received primed B cells from wt mice, but not from
LT-/- mice (Fig. 5),
confirming that the LT-/- environment does not support
the formation of memory B cells. Of interest, functional memory T cells
were recovered from both wt and LT-/- donors. Thus,
the altered microenvironment of LT-/- mice retains the
ability to support maturation of T cells to effective memory function.
The methods we have employed may not detect modest or partial
impairments of memory T cell formation in LT-/- mice.
Such potential memory T cell defects might be revealed by adoptive
transfer of smaller numbers of T cells, or by immunization with more
limiting doses of Ag. Nevertheless, our data do show that the tissue
requirements for the formation of memory B cells and memory T cells are
substantially different.
|
-/- microenvironment
GC, with their prominent clusters of FDC and scattered Ag-specific
T cells, are thought to provide primary venues for the formation of
memory B cells (13, 14, 19, 22). The GC represent a
dynamic microenvironment in which B cells differentiate first into
rapidly proliferating PNA+ cells that then move toward the
FDC clusters where further maturation takes place (13, 14, 23). The FDC are thought to play a central role in GC function,
with Ag deposited on their surfaces primarily in the form of
complement-coated immune complexes serving to select B cells with high
affinity Ag receptors. In the course of this selection process,
PNA+ GC cells are thought to be converted to the precursors
of both IgG-producing B cells and long-lived memory B cells. To
investigate further the nature of the disturbed B cell memory function
in the LT-/- mice, we examined the roles of LT and
the lymphoid tissue microenvironment in the formation and function of
GC-like clusters of PNA+ cells. Wt or
LT-/- mice were sublethally irradiated, reconstituted
with splenocytes from wt donors, and immediately immunized with an i.v.
infusion of 108 SRBC. Ten days after transfer and
immunization, sections of spleen were stained with PNA (Fig. 6). Both wt and LT-/-
recipients could support the development of clusters of
PNA+ cells in response to Ag, although the sizes and number
of the clusters generally appeared to be reduced in the
LT-/- recipients. Importantly, no clusters of
PNA+ cells were detected in LT-/-
recipients that received splenocytes from LT-/- donors
(data not shown and 8). Thus, the development of the
PNA+ cells was dependent on transferred LT-expressing
splenocytes. The PNA+ clusters that were produced by wt
splenocytes 10 days after transfer into the disturbed microenvironment
of LT-/- recipients were not associated with
detectable clusters of FDC (data not shown and 8) and were unable
to support the development of a detectable Ag-specific serum IgG
response (Fig. 2
and 8). In addition, the clusters of
PNA+ cells that were found in LT-/-
recipients of wt splenocytes showed no association with apoptotic
selection (Fig. 6). Wt SRBC-immunized mice that received wt splenocytes
showed prevalent clusters of TUNEL+ cells associated with
GC. No clusters of TUNEL+ cells were detected in the white
pulp of SRBC-immunized LT-/- mice that had been
reconstituted with wt donor splenocytes. Apoptosis of GC cells is
thought to represent deletion of B cells that express low affinity Abs.
This deletion is thought to correlate with the selection of B cells
with high affinity and their conversion into memory cells (14, 15, 19). Our data indicate that the LT-/-
microenvironment characterized by lack of FDC clusters can support the
differentiation of Ag-stimulated B cells into PNA+ GC-like
cells, but fails to support either affinity-based selection of these
cells or their further differentiation into either IgG producing cells
or into memory B cells.
|
-/-
mice is unable to support the generation of memory B cells from naive
progenitors. This LT-/- microenvironment is also
unable to support the conversion of preformed wt memory B cells
(produced in a wt environment) into Ag-producing cells that express a
mature memory response. At least three characteristics of the normal
spleen white pulp fail to form properly in LT-/- mice
(1, 2, 7, 21): 1) the segregated T cell-predominant
periarteriolar lymphoid sheath (PALS), 2) the segregated B
cell-predominant marginal sinus structure with MAdCAM-1+
(mucosal addressin cell adhesion molecule-1+) vascular
endothelium and MOMA-1+ metallophilic macrophage
components, and 3) primary B cell follicles with clusters of FDC.
Cooperative interactions between T and B lymphocytes are required for
the formation of functional GC and high affinity isotype-switched Ig
responses. Cooperative B/T interactions have also been suggested to be
required for the generation of memory B cells (13, 22, 24). It is possible that segregation of T and B cells into
separate zones within the splenic white pulp facilitates properly
regulated interactions of Ag-specific T and B cells during both primary
and memory responses. LT-/- mice lack normal T and B
cell segregation (1, 8). This might be expected to impair
effective collaboration between the two cell populations and explain
the observed inability to form memory B cells during a primary response
or to productively activate memory B cells in a memory response;
however, several observations argue against a major role of
disorganization of T and B cell zones in the impaired memory responses.
First, 6 wk after lethally irradiated LT-/- mice had
been reconstituted with wt bone marrow, segregation of T and B cell
compartments remained incomplete but FDC clusters were restored
(8). Mice reconstituted in this fashion can generate GC
and secondary IgG responses similar in magnitude to wt mice. Second, in
preliminary studies, we detect successful generation of memory B cells
in LT-/- mice that have been reconstituted with wt
bone marrow in this fashion (data not shown). Thus, normal T cell/B
cell segregation appears not to be required for the memory response.
Therefore, we speculate, that LT-dependent disturbances in the
primary B cell follicle structure are more likely to underlie the
disturbances of the memory response.
Consistent with this hypothesis, the generation of memory T cells
appears to require a microenvironment distinct from that required for
the formation of memory B cells. Memory T cells can be formed
effectively in LT-/- mice, with GC and FDC appearing
not to be required for the generation of memory T cells. FDC clusters,
on the other hand, are thought to play critical roles in the formation
of B cell follicles and in the presentation of Ag to activated B cells
(13, 14). We suggest that the lack of FDC clusters in
LT-/- mice primarily underlies the failure to generate
memory B cells and also to support the response of passively
transferred memory B cells to rechallenge. Consistent with this
hypothesis, our preliminary data suggest that LT-/-
mice fail to manifest affinity maturation following repeated challenge
with hapten in the absence of adjuvant (data not shown). Generally, a
lack of somatic mutation correlates tightly with failure to elaborate
memory B cells (14, 25).
The generation of the B cell arm of the memory response requires the
induction of memory B cells as well as their maintenance. The
experiments described here cannot distinguish between failure of the
initial differentiation of activated B cells into B cells of the memory
phenotype and failure to sustain the survival of these cells after Ag
is catabolized and cleared. Additional experiments in which exposure to
Ag is sustained continuously will be required to discriminate between
these potential mechanisms of the failure to generate transferrable
memory B cells in the LT-/- mice.
In addition, the failure of memory B cells induced in a wt environment
to express a memory IgG response after adoptive transfer into
sublethally irradiated LT-/- recipients could be based
on several potential mechanisms. Expression of a memory response by
adoptive transferred cells is contingent first on their survival in the
recipient mice. Although we know that similar numbers of donor B cells
are present in the spleen of recipient wt and LT-/-
mice after adoptive transfer (data not shown), the methods used here
cannot measure specifically the survival of memory cells. Assuming
survival of memory B cells after adoptive transfer, the expression of
the memory IgG response requires activation of these cells by Ag, and
then their conversion into IgG secreting cells. Our observation that
primed wt cells can support the Ag-dependent formation of clusters of
PNA+ cells after adoptive transfer into LT-/-
recipients suggests that Ag recognition does occur in this setting, and
that at least one key defect in the LT-deficient microenvironment
may be in conversion from activated, proliferating B cells to
Ab-secreting cells. Thus, the failure to express B cell memory in this
environment may represent a general failure of maturation of
proliferating, isotype-switched cells to Ab-secreting B cells and
plasma cells.
GC contain significant numbers of Ag-specific T cells, and these cells
appear to be essential for GC physiology, supporting somatic mutation
and affinity maturation of GC B cells (12, 13, 22, 26).
However, it has not been previously determined whether GC are required
for the generation of T cell memory. Our data indicate that generation
and subsequent activation of memory T cells and memory B cells require
different environmental elements. In contrast to memory B cells, memory
T cells can be generated in easily detectable numbers without the
formation of GC and FDC (Fig. 5).
Our data support the emerging concept that members of the TNF
ligand/receptor family play critical roles not only in the
establishment of normal lymphoid tissue structure, but also in the
cellular interactions that occur characteristically within these
tissues. In addition to the LTß receptor, several other members of
the TNF receptor family, including OX40, the nerve growth factor
receptor (NGFR), CD40, and type I TNF receptor (5, 27) are
required for different steps of the memory IgG response. OX40 and CD40
on B cells interact with their ligands on the surfaces of T cells, with
these interactions prominent during the generation of B cell memory
responses and crucial for the development of high affinity IgG
responses (5, 24). Signaling via the NGFR delivers
autocrine signals supporting the survival of memory B cells
(28). In these interactions, the B cell expresses the TNF
receptor family member and receives signals primarily from T cells to
support the Ab response. In contrast, B cells do not express the
LTßR. Instead, B cells express LT ligands (LT3 and
LT1ß2). Via these ligands, B cells signal
LTßR-bearing cells to support the development and maintenance of the
lymphoid tissue structure that is required for mature B cell responses
(11). The data presented here indicate that this
LT-dependent microenvironment is required both for the generation of
memory B cells and for their proper activation by recall Ag. These
results underscore the central role of the NGFR/TNF receptor family in
the generation of the memory response.
In summary, this study has demonstrated that LT-/-
mice show defective generation of memory B cells in response to
T-dependent Ags. Furthermore, they cannot support LT-expressing
memory B cells to express memory responses to recall Ags.
LT-expressing B cells, 10 days after they have been transferred into
LT-/- mice, can be activated by Ag to form clusters of
PNA+ cells; however, these cells do not form IgG-producing
cells. Unlike T cells that can develop memory responses in
LT-/- mice, B cell memory requires LT-dependent
structures, with either clusters of FDC or structures closely linked to
the expression of FDC being centrally required for the development of
this response. Our studies clearly separate the requirements for the
formation of memory T and memory B cells and establish lymphotoxin as a
member of the growing group of TNF family ligands that are required for
the maturation of the B cell response and the development of B cell
memory.
|
Footnotes |
|---|
2 Current address: Department of Pathology, University of
Chicago, Chicago, IL 60637-1470.
3 Address correspondence and reprint requests to Dr. David D. Chaplin, Division of Allergy and Immunology, Department of Internal Medicine, Washington University School of Medicine, 4566 Scott Avenue, Box 8122, St. Louis, MO 63110. E-mail address:
4 Abbreviations used in this paper: LT, lymphotoxin; LTßR, LT-ß receptor; FDC, follicular dendritic cell; GC, germinal center; KLH, keyhold limpet hemocyanin; wt, wild type; NP, nitrophenyl; PNA, peanut agglutinin; BCR, B cell Ag receptor, RU, relative units; AP, alkaline phosphatase.
Received for publication June 28, 1999. Accepted for publication December 22, 1999.
|
References |
|---|
|
TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
|---|
-deficient mice: effects on secondary lymphoid organ development and humoral immune responsiveness. J. Immunol. 155:1685.[Abstract]
/ß complex is required for the development of peripheral lymphoid organs. J. Exp. Med. 184:1999. supports development of splenic follicular structure that is required for IgG responses. J. Exp. Med. 185:2111. and ß revealed in lymphotoxin ß-deficient mice. Immunity 6:491.[Medline]
-dependent fashion. J. Exp. Med. 187:1009. (LT) deficient mice. Nature 382:462.[Medline]
-deficient and TNF receptor-I-deficient mice define developmental and functional characteristics of germinal centers. Immunol. Rev. 156:137.[Medline]
This article has been cited by other articles:
|
|
 |
|
  V. Bekiaris, F. Gaspal, M.-Y. Kim, D. R. Withers, F. M. McConnell, G. Anderson, and P. J. L. Lane CD30 Is Required for CCL21 Expression and CD4 T Cell Recruitment in the Absence of Lymphotoxin Signals J. Immunol., April 15, 2009; 182(8): 4771 - 4775. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Heikenwalder, M. Prinz, N. Zeller, K. S. Lang, T. Junt, S. Rossi, A. Tumanov, H. Schmidt, J. Priller, L. Flatz, et al. Overexpression of Lymphotoxin in T Cells Induces Fulminant Thymic Involution Am. J. Pathol., June 1, 2008; 172(6): 1555 - 1570. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M.-Y. Kim, F. M. McConnell, F. M. C. Gaspal, A. White, S. H. Glanville, V. Bekiaris, L. S. K. Walker, J. Caamano, E. Jenkinson, G. Anderson, et al. Function of CD4+CD3- cells in relation to B- and T-zone stroma in spleen Blood, February 15, 2007; 109(4): 1602 - 1610. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. Moreira and J. Faro Modelling Two Possible Mechanisms for the Regulation of the Germinal Center Dynamics J. Immunol., September 15, 2006; 177(6): 3705 - 3710. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. L. Wolniak, R. J. Noelle, and T. J. Waldschmidt Characterization of (4-Hydroxy-3-Nitrophenyl)Acetyl (NP)-Specific Germinal Center B Cells and Antigen-Binding B220- Cells after Primary NP Challenge in Mice J. Immunol., August 15, 2006; 177(4): 2072 - 2079. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. Y. Lee, E.-M. Ko, S.-H. Kim, D.-I. Jeoung, and J. Choe Human Follicular Dendritic Cells Express Prostacyclin Synthase: A Novel Mechanism to Control T Cell Numbers in the Germinal Center J. Immunol., August 1, 2005; 175(3): 1658 - 1664. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.-M. Park, S. Kim, J.-S. Choi, D.-Y. Hur, W.-J. Lee, M.-S. Lee, J. Choe, and T. H. Lee TGF-{beta} Inhibits Fas-Mediated Apoptosis of a Follicular Dendritic Cell Line by Down-Regulating the Expression of Fas and Caspase-8: Counteracting Role of TGF-{beta} on TNF Sensitization of Fas-Mediated Apoptosis J. Immunol., May 15, 2005; 174(10): 6169 - 6175. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Wakui, J. Kim, E. J. Butfiloski, L. Morel, and E. S. Sobel Genetic Dissection of Lupus Pathogenesis: Sle3/5 Impacts IgH CDR3 Sequences, Somatic Mutations, and Receptor Editing J. Immunol., December 15, 2004; 173(12): 7368 - 7376. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Manser Textbook Germinal Centers? J. Immunol., March 15, 2004; 172(6): 3369 - 3375. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. J. Hebeis, K. Klenovsek, P. Rohwer, U. Ritter, A. Schneider, M. Mach, and T. H. Winkler Activation of Virus-specific Memory B Cells in the Absence of T Cell Help J. Exp. Med., February 17, 2004; 199(4): 593 - 602. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z. SM. Rahman, S. P. Rao, S. L. Kalled, and T. Manser Normal Induction but Attenuated Progression of Germinal Center Responses in BAFF and BAFF-R Signaling-Deficient Mice J. Exp. Med., October 20, 2003; 198(8): 1157 - 1169. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. E. Lund, S. Partida-Sanchez, B. O. Lee, K. L. Kusser, L. Hartson, R. J. Hogan, D. L. Woodland, and T. D. Randall Lymphotoxin-{alpha}-Deficient Mice Make Delayed, But Effective, T and B Cell Responses to Influenza J. Immunol., November 1, 2002; 169(9): 5236 - 5243. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. J. Marshall, Q. Du, K. E. Draves, Y. Shikishima, K. T. HayGlass, and E. A. Clark FDC-SP, a Novel Secreted Protein Expressed by Follicular Dendritic Cells J. Immunol., September 1, 2002; 169(5): 2381 - 2389. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. G. Hannum, A. M. Haberman, S. M. Anderson, and M. J. Shlomchik Germinal Center Initiation, Variable Gene Region Hypermutation, and Mutant B Cell Selection without Detectable Immune Complexes on Follicular Dendritic Cells J. Exp. Med., October 2, 2000; 192(7): 931 - 942. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
;
LT
) were immunized i.p. on day 0 with
108 SRBC without adjuvant (A) or with IFA
(B) and given a boost with the same dose without adjuvant 3
mo later. Serum was collected 10 days after the initial immunization
(Primary) and 5 days after the booster immunization (Memory).
SRBC-specific IgG was measured by ELISA as described in Materials
and Methods. Data shown represent the means ± SEM of
triplicate determinations from three to five mice. Similar results were
obtained in a replicate experiment.
, splenocytes from naive donors transferred to a
LT
, splenocytes from primed
wt donors transferred to a LT
