By
From the * Section of Immunobiology and § Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520-8011
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Abstract |
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Follicular dendritic cell networks are said to be pivotal to both the formation of germinal centers (GCs) and their functions in generating antigen-specific antibody affinity maturation and B
cell memory. We report that lymphotoxin -deficient mice form GC cell clusters in the gross
anatomical location expected of GCs, despite the complete absence of follicular dendritic cell networks. Furthermore, antigen-specific GC generation was at first relatively normal, but these
GCs then rapidly regressed and GC-phase antibody affinity maturation was reduced. Lymphotoxin
-deficient mice also showed substantial B cell memory in their mesenteric lymph
nodes. This memory antibody response was of relatively low affinity for antigen at week 4 after
challenge, but by week 10 after challenge was comparable to wild-type, indicating that affinity maturation had failed in the GC phase but developed later.
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Introduction |
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Follicular dendritic cells (FDCs)1 are characterized by their location within B cell follicles and their extensive dendritic process networks, which retain antigen-Ig complexes via both complement receptors and Ig Fc receptors (for reviews, see references 1). Some antigen retained by FDCs is held within convolutions of the dendritic process network for periods of >1 yr and is thought to play an important role in the maintenance of long-term B cell memory. It is perhaps these same processes that have gained FDCs notoriety as a reservoir of HIV (5).
FDCs also give up some of their retained antigen much earlier, during the germinal center (GC) reaction. GCs are highly specialized structures that develop within primary B cell follicles upon antigenic challenge (9, 10). Antigen-specific B cells within GCs undergo somatic hypermutation of their antigen-binding receptor in a process of antibody affinity maturation (9). GC B cells are intimately associated with FDC dendritic processes, and antigen provided by FDC networks is thought to be critical for the selection of higher affinity clones within GCs and subsequent memory B cell formation (1, 12). FDCs also provide other soluble and contact-dependent signals which promote GC B cell viability, proliferation, and chemotactic responsiveness (4, 13). It has also been asserted that FDCs may be the "nucleating force" for the initial formation of GCs within the specific anatomical location they occupy (4).
Lymphotoxin (LT) is an immediate member of the TNF
family, first identified by virtue of its ability to anchor LT
(TNF-
) to the surface of T cell hybridomas (14, 15). Both
LT
and LT
are now known to be produced by activated
B cells, activated T cells, and NK cells (16, 17). Effects of
LT
R engagement include integrin upregulation (18), cytotoxicity (19, 20), and induction of chemokine production
(21). Most notably, lt
/
mice, lt
/
mice, and lt
r
/
mice lack peripheral LNs and Peyer's patches, and have a
disorganized splenic architecture with almost complete loss
of GCs and FDC networks (22). Having said this, most
lt
/
mice still have mesenteric (M)LNs (26, 27, 30). Furthermore, the MLNs still form GC-like cell clusters in rudimentary B cell follicles despite the apparent lack of FDC networks (26, 30). Similar observations have been made in the
spleen of lt
/
mice and lt
r
/
mice (27), although
these studies also found residual FDC-like cells. Although
not yet proven, the latter may be FDC precursors or immature FDCs but equally so might represent GC dendritic cells (GCDCs), which are distinct from FDCs and have so far
been described in human tonsils (31) but not in mice.
Therefore, lt/
mouse MLNs appeared to present a
unique model of in vivo GC reaction in the absence of FDC
networks. Although other studies have suggested that GCs can
form in the absence of antigen trapping on FDCs (32), an
FDC-less mouse model was unprecedented and would further
studies of GC reaction. Lt
/
mice might also facilitate the
identification and evaluation of GCDCs. Finally, knowledge
of the consequences of a lack of FDC networks will also be essential in assessing the potential utility of causing FDC regression. That is, it was recently reported that administration of
soluble LT
R to adult mice results in temporary regression of
FDC networks (33). Thus, there is now the prospect of being
able to eliminate FDC networks in circumstances in which this might be considered likely to yield therapeutic benefit. For example, loss of FDC networks during triple drug therapy
might eliminate this important HIV reservoir.
With all of this in mind, this study reports the dynamics of
GC reaction and B cell memory formation in lt/
mice.
Initiation of antigen-specific GCs in the MLNs of lt
/
mice was relatively normal, but GC B cell numbers subsequently fell dramatically. GC B cells were clustered in the
anatomical locations expected of GCs, indicating that this
process is not critically dependent on FDC networks. Lt
/
mice also generated substantial B cell memory despite defective GC processes, albeit of relatively low affinity. Some antigen-specific antibody affinity maturation did occur and was
even more apparent in the memory response, suggesting that
somatic hypermutation had occurred. This is in agreement
with other studies where somatic hypermutation was evident, even in the complete absence of GCs (24, 34).
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Materials and Methods |
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Reagents.
The protein G-Sepharose column-purified antibodies anti-Animals and Animal Challenge.
Cd40lMLN Histology.
MLNs were frozen in Tissue-Tek OCT compound (VWR Scientific) using a dry-ice/methylbutane bath and stored atMLN Cell Preparation.
MLNs were harvested into 0.5 ml cold digest buffer in a 4-well plate (Nunc) on ice. Digest buffer was Bruff's medium with 5% FCS, 0.1 mg/ml collagenase type IV (Sigma), and 0.1 mg/ml deoxyribonuclease type I (Sigma). Bruff's medium is Click's medium (Irvine Scientific) supplemented with 40 mM L-glutamine, 60 µM 2-ME, 0.7 mM sodium bicarbonate, and 58 mg/l gentamycin. The capsule of each MLN was torn and teased open with 27-gauge needles before incubating plates at 37°C for 30 min. Capsules were then further disrupted to release MLN cells by pipetting. The cell suspension was made up to 10 ml with Bruff's/5% FCS, filtered through 0.1-mm nylon mesh (Small Parts, Inc.), and centrifuged at 800 revolutions/min in a bench-top centrifuge at 4°C for 5 min. Finally, cells were resuspended in 2 ml Bruff's/5% FCS for fluorocytometry or antigen-specific antibody determination as described below.Fluorocytometry.
MLN cells were prepared as described above, and aliquots of 106 cells were resuspended into 0.2 ml PBS/1% FCS supplemented with 5 µg/ml FcBlock (PharMingen). Samples were left on ice for 30 min before primary antibodies were added, and were then left on ice in the dark for an additional 1 h. Samples were washed by being made up to 1.2 ml with PBS/1% FCS before centrifugation at 800 revolutions/min at 4°C for 5 min. Secondary antibody incubation and rewashing were done as above. Four-color fluorocytometry used a FACSCalibur® with argon and helium-neon lasers (Becton Dickinson). Data from 2.5 × 105 events were analyzed with CellQuest software by first gating on lymphocytes/lymphoblasts, based on forward and side scatter. PharMingen antibodies used included anti-CD4-allophycocyanin (APC; L3T4, RM4-5), anti-CD8MLN Antigen-specific Antibody Determination.
Antigen-specific antibody determination was done essentially as described by Kelly et al. (36), using antibody-secreting cells directly in an ELISA. Maxisorp 96-well plates (Nunc) were coated at 4°C for 16-20 h with 10 µg/ml NP2BSA or NP15BSA in PBS, 0.1 ml aliquot per well. Plates were then washed four times with PBS before blocking with PBS/1% FCS for 2-3 h. Meanwhile, MLN cells were prepared at 107 cells per ml of Bruff's/5% FCS as described above. MLN cell aliquots of 0.1 ml were then plated in wells and cultured at 37°C for 5 h alongside a twofold serial dilution of serum from a standard hyperimmunized wild-type mouse (see above; serum dilution was with PBS/1% FCS). Subsequent washes were with PBS/0.05% Tween 20. Ig detection used isotype-specific alkaline phosphatase-conjugated reagents and pNPP substrate as described (Southern Biotechnology Associates). OD405 was determined with a microplate reader (model 550; BioRad). Typically, a 1:12,800 dilution of hyperimmunized wild-type mouse serum gave an OD405 of ~2.7 and 1.9 above background for IgG1 detected with NP2BSA and NP15BSA, respectively. The lower limit of detection was typically at a serum dilution of 1:1,638,400, giving an OD405 of ~0.05-0.10 above background. The anti-NP-specific antibody titer of samples was expressed as relative units, representing the reciprocal of the standard serum dilution giving the same OD405 as the sample. Calculations were by four-parameter analysis using Microplate Manager III software (BioRad).Serum Antigen-specific Antibody Determination.
Blood was harvested from mice for serum by cardiac puncture at the time of culling. Maxisorp plates were coated with NP2BSA or NP15BSA, blocked, and washed as described above. Serum aliquots of 0.1 ml (1:105 in PBS/1% FCS) were applied to wells for 1 h alongside a twofold serial dilution of serum from a hyperimmunized wild-type mouse (see above; serum dilution was with PBS/1% FCS). Antigen-specific Ig determination then proceeded as described in the previous section. ![]() |
Results |
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We showed previously that lt/
mice form PNA-binding, IgD
B220+ cell clusters in B cell areas of their MLNs
(26, 30), characteristic of GCs (37). Although IgD+ B cells
were also found to infiltrate T cell areas, the GC-like B cell
clusters were within IgD+ B cell follicles at the periphery of
MLNs, as in wild-type mouse MLNs (26, 30). These observations were extended here by staining for CD24. Primary follicle B cells are CD24+, but GC B cells are CD24hi
(38-40; Fig. 1 A). This also appeared to be true of lt
/
GC B cell clusters (Fig. 1 B). Again, the lt
/
GC B cells
were clustered within the location expected of wild-type GCs.
|
Immunohistology with anti-complement receptor 1 (CR-1)
and FDC-M1 suggested that lt/
mice lack FDCs (26).
We subsequently showed that although wild-type mouse
MLNs have very large amounts of IgM immune complex
on FDCs, lt
/
mouse MLNs did not have any such deposits (30). However, despite all of the above, it is possible
that Ig Fc receptor-bearing (FcR+) FDCs (3, 4, 9) constitute a distinct subset of FDCs which are negative for both
FDC-M1 and CR-1. It is also conceivable that antigen-Ig
complexes retained on such FcR+ FDCs may support GC
reaction in the absence of CR-1+ FDCs. In potential support of this, mice deficient in CR-1 still possess GCs (41,
42), albeit somewhat smaller than wild-type.
Therefore, an evaluation was made as to whether or not
lt/
mice possess FcR+ FDC networks by staining for
CD23 (43). This revealed that lt
/
mice completely lack
FcR+ FDC networks (Fig. 1 D), and that the lt
/
GC B
cell clusters were CD23
compared with the surrounding
follicle B cells, as expected of wild-type GC B cells (9; Fig.
1 C). FDC networks clearly stained very strongly for CD23
in wild-type mouse MLNs (Fig. 1 C), and were observed
in every B cell follicle regardless of whether or not there
was an ongoing GC reaction. Some discrete CD23lo cells
were seen in the lt
/
mouse MLN CD23
IgD
GC cell
clusters (Fig. 1 D). These cells most likely represent GC B
cells that have not yet fully downregulated CD23, but it is
conceivable that they represent immature FDCs or the
mouse equivalent of GCDCs (31).
Finally, the lack of staining by FDC-M2 further emphasized the complete absence of FDC networks in the MLNs
of lt/
mice (Fig. 1 F). Although FDC-M2 has not been
fully characterized, it is a useful marker of FDC networks
(Fig. 1 E). Like CD23, FDC-M2 staining was observed in
every wild-type mouse MLN B cell follicle regardless of
whether or not there was an ongoing GC reaction (data
not shown). As with CD23, a low level of FDC-M2 staining may well mark immature FDCs or another cell type
such as the mouse equivalent of GCDCs (31). Neither immature FDCs nor mouse GCDCs have yet been defined.
Four-color fluorocytometry was adopted
in order to study further the GC reaction in lt/
mouse
MLNs (see Materials and Methods). Most non-GC B cells
were excluded as "lineage"-positive (Lin+) cells, using a
combination of antibodies against CD4, CD8
, CD90.2,
IgD, and CD11b (Fig. 2). Markers to discriminate GC B cells included PNA binding (37, 38), anti-CD24 (38),
and GL7 (44). PNA binding and CD24 are increased on
GC B cells, whereas GL7 recognizes an activation antigen
found on GC B cells but not naive or memory B cells. Fluorocytometry of MLNs is simple compared with spleen
due to the relative lack of immature B cells, granuloid cells,
and erythroid cells. After excluding nonlymphoid particles
of low forward scatter (data not shown), most Lin
cells in
MLNs are PNAhiCD24hi (data not shown). This is demonstrated in Fig. 2 with MLNs 12 d after intraperitoneal challenge with CG adsorbed to alum.
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As expected, both T cell-less mice and cd40l/
mice fail
to generate GC B cells (Fig. 2), most clearly demonstrated
with GL7. Both wild-type mice and lt
/
mice revealed
substantial levels of Lin
GL7+ cells in their MLNs, although lt
/
mice consistently had lower levels than
wild-type mice (see also below). Lin
GL7+ cells represented about half of all Lin
PNAhiCD24hi cells (Fig. 2) and
were CD45R+ (data not shown; see below). Lin
cells include non-GC B cells such as antibody-secreting cells and
memory B cells, which would be GL7
.
The levels of LinGL7+ GC B cells at day 12 after challenge (Fig. 2) were much higher than those in unchallenged mice, and were similar to those observed in the
spleen with the same antigenic challenge (45). Without intraperitoneal challenge, the levels of Lin
GL7+ cells were
0.3-0.7% of total MLN cells in both wild-type mice and
lt
/
mice (data not shown; see below), consistent with
levels in the spleen of unchallenged wild-type mice (45).
Thus, intraperitoneal challenge in alum is an effective
means of inducing GC B cell generation in the MLNs of
both wild-type mice and lt
/
mice.
The
hapten NP has been used extensively in GC studies (40, 45-
48). The primary response to NP is dominated by antibodies bearing a 1 light chain, recognized by the antiidiotype antibody Ls136 (39, 49). NP-specific antibody-bearing cells
can also be followed by their capacity to bind NP-conjugated
PE (NP20PE). NP also provides a means for evaluating anti-NP-specific antibody affinity maturation (described below).
Mice were challenged intraperitoneally with either CG
or NP-haptenated CG (NP13CG), and MLNs were examined for NP-specific response 8 d later. Both wild-type mice and lt/
mice showed massive numbers of
1+
NP20PE-binding MLN cells in response to NP13CG
(Fig. 3). The percentage of CD45R+ cells that were
1+
NP20PE-binding were 66.1 ± 18.2 and 76.6 ± 17.2 for
wild-type mouse MLNs and lt
/
mouse MLNs, respectively (n = 6 each), compared with <0.1% in unchallenged
mice. As expected (46), CG itself did not elicit any significant levels of
1+ NP20PE-binding cells (data not shown).
Also, T cell-less mice and cd40l
/
mice did not show significant levels of
1+ NP20PE-binding cells (Fig. 3).
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NP-specific GC B cells were also evident in MLNs from
both wild-type mice and lt/
mice at day 8 after challenge (Fig. 4), defined as the Lin
GL7+ subset (see Fig. 2).
Both the fraction of lt
/
GC B cells that were NP20PE-binding and the intensity of NP20PE-binding appeared to
be relatively normal (Fig. 4). The response of wild-type mice and lt
/
mice was then followed from day 8 to day
24 after challenge (Fig. 5). As early as day 12 after challenge, lt
/
mouse MLN
1+ GC B cells were greatly
reduced compared with wild-type, and even fewer were
NP20PE-binders (Fig. 5). Having said this, the rate of decline of lt
/
GC B cells was not sustained at day 16 after
challenge. Instead, Lin
GL7+
1+ lt
/
GC B cell levels
appeared to plateau such that they were comparable to
wild-type at day 20 after challenge before then falling again by day 24 (Fig. 5). This pattern was reflected among
Lin
GL7
1+ non-GC cells.
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Both
lt/
mice and wild-type mice showed anti-NP antibody secretion among MLN cells at day 6 after challenge with 50 µg
NP13CG adsorbed to alum (Fig. 6). IgA and IgG2a were not
detected at all (data not shown). Clearly, IgG1 secretion by
lt
/
mouse MLN cells (average relative units = 0.91) was
much lower than wild-type (average = 6.03), perhaps indicating a lack of T cell and/or dendritic cell help. Nonetheless, this
level of IgG1 secretion by lt
/
mouse MLNs was ~20-fold
higher than the lower limit of detection. Also, a primary challenge with 0.2 mg NP13CG in PBS alone did not result in significant anti-NP antibody secretion by either wild-type or
lt
/
mouse MLN cells at day 6 after challenge (data not
shown). Thus, the response to NP13CG in PBS was a good indicator of memory generated as a result of primary challenge
with NP13CG adsorbed to alum (see below).
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To determine whether or not humoral memory was generated, mice were challenged as before and then rechallenged at various times with either 0.2 mg NP13CG in PBS without alum or PBS alone. 6 d later, anti-NP antibody was determined among MLN cells and in serum. Both the level of anti-NP antibody and the relative affinity for NP were determined by ELISA using two different BSA substrates. Total and relatively high affinity anti-NP antibody were determined with densely (NP15BSA) and sparsely (NP2BSA) haptenated BSA, respectively. As GC reaction proceeds, a greater proportion of anti-NP antibody becomes detectable with NP2BSA. The principal of this approach has been used in several other studies (24, 26, 34), and was recently validated with mAbs of various affinity for NP (45).
When given a secondary challenge and harvested at week
4, lt/
mouse MLNs appeared to secrete more anti-NP
antibody than wild-type mouse MLNs (Fig. 7), implying
that lt
/
mice had generated greater humoral memory
than wild-type mice. This pattern was not so apparent in serum (Fig. 7). Nonetheless, lt
/
mice clearly showed memory even in their serum. The substantial memory response
was still evident among lt
/
mouse MLNs at week 10 after challenge but was less evident in serum (Fig. 7).
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Both wild-type mouse MLNs and lt/
mouse MLNs
showed affinity maturation in the post-GC phase between
weeks 4 and 10 after challenge (Fig. 7). Thus, the relatively
low NP2/NP15 ratio in lt
/
mouse MLNs at week 4 after
challenge was almost normal at week 10. Again, this observation in MLNs was not obvious in serum (Fig. 7). At both
weeks 4 and 10 after challenge, lt
/
mice showed a
lower NP2/NP15 ratio in their serum than wild-type mice,
indicating a defect in GC affinity maturation processes.
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Discussion |
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Numerous lines of evidence have now shown that lt/
mice do not have FDC networks (26, 27, 30, and this
study). In addition, MacKay and Browning (33) have very
recently shown that administration of soluble LT
R to
adult mice causes regression of FDC networks and dissipation of the antigen they were retaining. Clearly however, lt
/
mice might still have FDC precursors. Indeed, irradiated adult lt
/
mice generate mature FDC networks of
lt
/
origin upon reconstitution with wild-type bone
marrow (53), and equivalent results have been obtained
with lt
/
mice (data not shown). The location of FDC
precursors in lt
/
mice is at present unknown, and it is
conceivable that they will be found within B cell follicles
and GCs even though they fail to develop into mature
FDC networks. Others have shown that lt
/
mice and
lt
r
/
mice do have small numbers of discrete FDC-M2+
cells in the spleen (27, 28), leading to the suggestion that these cells may represent FDC precursors or immature
FDCs. However, there is no evidence that FDC-M2 is completely specific to FDCs, although it is a useful marker of
FDC networks. Unlike FDC-M2, FDC-M1 has been well
characterized and is considered to be relatively FDC specific,
but even this marker also stains tingible body macrophages (which are found in GCs) and some endothelial cells (54).
The origin of FDCs is somewhat controversial (for a review, see reference 55) and will undoubtedly be further
complicated by the fact that mice may also prove to have
GCDCs (31), which might be the FDC-M2+ cells seen by
others. Regardless of whether or not lt/
mice have
FDC precursors, the roles FDCs are said to fulfill largely
rely on the extensive dendritic processes of FDC networks. Clearly, these structures are absent.
The absence of FDC networks presumably has indirect
as well as direct effects on GC reactions. For example, this
study has not considered GC T cells in lt/
mice. Other
defects clearly exist in lt
/
mouse MLNs, such as the B
cell infiltration of the T cell areas and reduced primary humoral response. What this study has attempted to do is
highlight the processes that occur despite the defects. Most
notably, lt
/
mice generate antigen-specific GC B cells
and class-switched memory B cell responses. Having said
this, antigen-specific lt
/
GC B cells decline rapidly at
times when wild-type GC B cell numbers are still relatively
high. GCs normally have a life span of a few weeks (10,
56). It has been argued that the regression of GCs begins at
a time when FDCs begin to bury their retained antigen
within membrane pockets, thereby ceasing to present antigen to GC B cells (56). The decline of lt
/
GC B cells in
the absence of FDC networks may be a premature execution of this process. On the other hand, lt
/
GC B cell
numbers did not decline further between days 12 and 16 after challenge but instead appeared to plateau until they finally decreased further between days 20 and 24. It is conceivable that the Lin
GL7+
1+ cell numbers were maintained by further generation of such cells from centroblasts.
Anti-NP memory and relative affinity maturation were
assessed at various times. The humoral memory response in
lt/
mouse MLNs was as great if not greater than that
seen in wild-type mouse MLNs (Fig. 7). Of course, this
may not be a direct reflection of the actual frequency of
NP-specific memory B cells. The relatively low level of
NP-specific non-GC B cells (Lin
1+ NP20PE-binding) in
lt
/
mouse MLNs late in the GC phase (Fig. 5) suggests
that lt
/
mouse MLNs produce significantly fewer memory B cells than wild-type mouse MLNs.
The degree of affinity maturation observed in serum
here was similar to that previously reported for lt/
mice, Lyn kinase (lyn)
/
mice, and lt
/
mice (24, 26,
34). Affinity maturation requires somatic hypermutation
and subsequent selection of higher affinity clones, suggesting that specific activated B cells had entered into a "GC B
cell program" despite the complete absence of GCs in lt
/
mice and lyn
/
mice. Indeed, somatic hypermutation is
evident in lt
/
mice and lyn
/
mice (24, 34), and there
is no reason to believe that this is not occurring in lt
/
mice. Certainly, somatic hypermutation is normally evident as early as day 7 after challenge (40, 45, 48, 57, 58) and, unlike lt
/
mice (24) and lyn
/
mice (34), lt
/
mice generate appreciable levels of antigen-specific GC B
cells upon challenge (this study).
Where and how are high-affinity mutants selected in the
absence of GCs? Takahashi et al. (45) recently described
evidence in support of a phenomenon best described as
"post-GC intraclonal competition." The average affinity of
anti-NP antibody from bone marrow antibody-secreting
cells continued to increased long after the GC reaction had
waned (45). Although clonal selection occurs independently in each GC and low-affinity B cells can survive the
selection process within GCs if high-affinity competitors are absent (45), post-GC affinity-driven selection processes effectively constitute "inter-GC selection." This concept is
supported by the study here, where the relative affinity of
anti-NP antibody at week 10 after challenge was about
twofold higher than that at week 4 after challenge (Fig. 7),
in the MLNs of wild-type mice and lt/
mice.
Thus, mutants with higher affinity for the antigen are
generated and selected even in lt/
mice, but the highest
possible affinity for antigen is not achieved in lt
/
mice
(Fig. 7), presumably because further rounds of somatic mutation cannot occur in the post-GC phase. Hence, despite
post-GC intraclonal competition, the benefit of the GCs is
as an environment in which repeated rounds of somatic
mutation and selection can occur rapidly in order to
achieve the highest possible affinity for antigen. Indeed, although substantial somatic hypermutation was observed in
lt
/
mice, the degree of mutation among V186.2 genes
was only about half that seen in wild-type mice (24).
It should also be borne in mind that the apparent degree
of affinity maturation by post-GC intraclonal competition
will vary substantially depending on the nature of the antigen. In some experimental instances, random mutations lead
to higher affinity clones at a relatively high frequency (59).
The NP hapten used here and by others (24, 34, 45) would
appear to be another such example, since a frequently occurring single point mutation in the V186.2 gene (24, 60) is associated with greatly increased affinity for NP (61). Thus, the
studies here showing defective NP-specific affinity maturation in lt/
mice can only be interpreted to suggest that
affinity maturation within GCs has failed in these mice.
Recirculating memory B cells migrate between secondary
lymphoid organ follicles where they can respond to antigen
held on FDC networks (62, 63). Marginal zone memory B
cells are said to be long-lived and non-recirculating (64),
and it is difficult to conceive of how their maintenance could
be dependent on antigen retained by FDC networks. As the
name suggests, marginal zone memory B cells have been best
characterized in the marginal zone of the spleen, where they
are seen to appear both immediately before and during GC
reactions (63). Equivalent areas are located on the inner
wall of the subcapsular sinus of LNs, and may be substantial
in MLNs (65). Further studies will be necessary to characterize the B cell memory in lt/
mice, including how the nature and dose of antigen might affect memory maintenance
besides memory generation per se.
In conclusion, this study has considered GC reactions in
the MLNs of lt/
mice and found that both GC and B
cell memory are formed despite the complete absence of
FDC networks. However, antigen-specific antibody affinity maturation is defective. This study of the MLNs of lt
/
mice serves as a model for the consequences of administration of soluble LT
R with respect to LNs. The spleen of
lt
/
mice was not studied here because it is more disorganized than the MLNs (26, 27, 30). The fact that anti-NP
memory at week 10 was much less apparent in serum than
in MLNs (Fig. 7) may well be a reflection of the fact that
serum antibody levels are dominated by the spleen and
spleen-derived bone marrow antibody-secreting cells. Having said this, even the spleen of lt
/
mice generates some
PNA-binding GC-like B cells (27).
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Footnotes |
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Address correspondence to Richard A. Flavell, Section of Immunobiology and Howard Hughes Medical Institute, 310 Cedar St., FMB412, Yale University School of Medicine, New Haven, CT 06520-8011. Phone: 203-737-2216; Fax: 203-785-7561; E-mail: richard.flavell{at}qm.yale.edu
Received for publication 1 December 1998 and in revised form 15 January 1999.
We are indebted to Garnett Kelsoe for Ls136 antibody, Marie Kosco-Vilbois for FDC-M2 antibody, Frank Wilson for technical assistance, Fran Manzo for clerical assistance, and Michiko Shimoda for invaluable advice and discussion.
This study was supported by the American Diabetes Association and the Howard Hughes Medical Institute. R.A. Flavell is an Investigator of the Howard Hughes Medical Institute.
Abbreviations used in this paper
APC, allophycocyanin;
CG, chicken
-globulin;
DIG, digoxigenin;
FDC, follicular dendritic cell;
GC, germinal
center;
GCDC, GC dendritic cell;
Lin, lineage;
LT, lymphotoxin;
MLN, mesenteric lymph node;
NP, (4-hydroxy-3-nitrophenyl)acetyl;
PNA, peanut agglutinin.
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References |
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1. | Klaus, G.G.B., J.H. Humphrey, A. Kunkl, and D.W. Dongworth. 1980. The follicular dendritic cell: its role in antigen presentation in the generation of immunological memory. Immunol. Rev. 53: 3-28 [Medline]. |
2. | Mandel, T.E., R.P. Phipps, A. Abbot, and J.G. Tew. 1980. The follicular dendritic cell: long term antigen retention during immunity. Immunol. Rev. 53: 29-59 [Medline]. |
3. | Schriever, F., and L.M. Nadler. 1992. The central role of follicular dendritic cells in lymphoid tissues. Adv. Immunol. 51: 243-284 [Medline]. |
4. | Tew, J.G., J. Wu, D. Qin, S. Helm, G.F. Burton, and A.K. Szakal. 1997. Follicular dendritic cells and presentation of antigen and costimulatory signals to B cells. Immunol. Rev. 156: 39-52 [Medline]. |
5. | Schrager, L.K., and A.S. Fauci. 1995. Trapped but still dangerous. Nature. 377: 680-681 [Medline]. |
6. | Heath, S.L., J.G. Tew, J.G. Tew, A.K. Szakal, and G.F. Burton. 1995. Follicular dendritic cells and human immunodeficiency virus infectivity. Nature. 377: 740-744 [Medline]. |
7. |
Haase, A.T.,
K. Henry,
M. Zupancic,
G. Sedgewick,
R.A. Faust,
H. Melroe,
W. Cavert,
K. Gebhard,
K. Staskus,
Z.Q. Zhang, et al
.
1996.
Quantitative image analysis of HIV-1 infection in lymphoid tissue.
Science.
274:
985-989
|
8. | Burton, G.F., A. Masuda, S.L. Heath, B.A. Smith, J.G. Tew, and A.K. Szakal. 1997. Follicular dendritic cells (FDC) in retroviral infection: host/pathogen perspective. Immunol. Rev. 156: 185-197 [Medline]. |
9. | MacLennan, I.C.M.. 1994. Germinal centers. Annu. Rev. Immunol. 12: 117-139 [Medline]. |
10. | Kelsoe, G.. 1996. In situ studies of the germinal center reaction. Adv. Immunol. 60: 267-288 . |
11. | Rajewsky, K.. 1996. Clonal selection and learning in the antibody system. Nature. 381: 751-758 [Medline]. |
12. | Kunkl, A., and G.G.B. Klaus. 1981. The generation of memory cells. I. Immunization with antigen-antibody complexes accelerates the development of B-memory cells, the formation of germinal centres and the maturation of antibody affinity in the secondary response. Immunology. 43: 371-378 [Medline]. |
13. | Lindhout, E., G. Koopman, S.T. Pals, and C. de Groot. 1997. Triple check for antigen specificity of B cells during germinal center reactions. Immunol. Today. 18: 573-577 [Medline]. |
14. |
Androlewicz, M.J.,
J.L. Browning, and
C.F. Ware.
1992.
Lymphotoxin is expressed as a heteromeric complex with a
distinct 33-kDa glycoprotein on the surface of an activated
human T cell hybridoma.
J. Biol. Chem.
267:
2542-2547
|
15. |
Browning, J.L.,
A. Ngam-ek,
P. Lawton,
J. DeMarinis,
R. Tizard,
E.P. Chow,
C. Hession,
B. O'Brine-Greco,
S.F. Foley, and
C.F. Ware.
1993.
Lymphotoxin ![]() |
16. | Paul, N.L., and N.H. Ruddle. 1988. Lymphotoxin. Annu. Rev. Immunol. 6: 407-438 [Medline]. |
17. | Ware, C.F., T.L. VanArsdale, P.D. Crowe, and J.L. Browning. 1995. The ligands and receptors of the lymphotoxin system. Curr. Top. Microbiol. Immunol. 198: 175-218 [Medline]. |
18. | Hochman, P.S., G.R. Majeau, F. Mackay, and J.L. Browning. 1996. Proinflammatory responses are efficiently induced by homotrimeric but not heterotrimeric lymphotoxin li- gands. J. Inflamm. 46: 220-234 . |
19. |
Browning, J.L.,
K. Miatkowski,
I. Sizing,
D. Griffiths,
M. Zafari,
C.D. Benjamin,
W. Meier, and
F. Mackay.
1996.
Signaling through the lymphotoxin ![]() |
20. |
MacKay, F.,
P.R. Bourdon,
D.A. Griffiths,
P. Lawton,
M. Zafari,
I.D. Sizing,
K. Miatkowski,
A. Ngam-ek,
C.D. Benjamin,
C. Hession, et al
.
1997.
Cytotoxic activities of recombinant soluble murine lymphotoxin-![]() ![]() ![]() |
21. |
Degli-Esposti, M.A.,
T. Davis-Smith,
W.S. Din,
P.J. Smolak,
R.G. Goodwin, and
C.A. Smith.
1997.
Activation of the
lymphotoxin ![]() |
22. | De Togni, P., J. Goellner, N.H. Ruddle, P.R. Streeter, A. Fick, S. Mariathasan, S.C. Smith, R. Carlson, L.P. Shornick, J. Strauss-Schoenberger, et al . 1994. Abnormal development of peripheral lymphoid organs in mice deficient in lymphotoxin. Science. 264: 703-707 [Medline]. |
23. |
Banks, T.A.,
B.T. Rouse,
M.K. Kerley,
P.J. Blair,
V.L. Godfrey,
N.A. Kuklin,
D.M. Bouley,
J. Thomas,
S. Kanangat, and
M.L. Mucenski.
1995.
Lymphotoxin-![]() |
24. |
Matsumoto, M.,
S.F. Lo,
C.J.L. Carruthers,
J. Min,
S. Mariathasan,
G. Huang,
D.R. Plas,
S.M. Martin,
R.S. Geha,
M.H. Nahm, and
D.D. Chaplin.
1996.
Affinity maturation without
germinal centers in lymphotoxin-![]() |
25. | Matsumoto, M., S. Mariathasan, M.H. Nahm, F. Baranyay, J.J. Peschon, and D.D. Chaplin. 1996. Role of lymphotoxin and the type I TNF receptor in the formation of germinal centers. Science. 271: 1289-1291 [Abstract]. |
26. |
Koni, P.A.,
R. Sacca,
P. Lawton,
J.L. Browning,
N.H. Ruddle, and
R.A. Flavell.
1997.
Distinct roles in lymphoid organogenesis for lymphotoxins ![]() ![]() ![]() |
27. |
Alimzhanov, M.B.,
D.V. Kuprash,
M.H. Kosco-Vilbois,
A. Luz,
R.L. Turetskaya,
A. Tarakhovsky,
K. Rajewsky,
S.A. Nedospasov, and
K. Pfeffer.
1997.
Abnormal development of
secondary lymphoid tissues in lymphotoxin ![]() |
28. |
Fütterer, A.,
K. Mink,
A. Luz,
M.H. Kosco-Vilbois, and
K. Pfeffer.
1998.
The lymphotoxin ![]() |
29. |
Alexopoulou, L.,
M. Pasparakis, and
G. Kollias.
1998.
Complementation of lymphotoxin ![]() |
30. |
Koni, P.A., and
R.A. Flavell.
1998.
A role for tumor necrosis
factor receptor type 1 in gut-associated lymphoid tissue development: genetic evidence of synergism with lymphotoxin
![]() |
31. | Grouard, G., I. Durand, L. Filgueira, L. Banchereau, and Y.-J. Liu. 1996. Dendritic cells capable of stimulating T cells in germinal centers. Nature. 384: 364-367 [Medline]. |
32. | Kroese, F.G.M., A.S. Wubbena, and P. Nieuwenhuis. 1986. Germinal centre formation and follicular antigen trapping in the spleen of lethally X-irradiated and reconstituted rats. Immunology. 57: 99-104 [Medline]. |
33. | MacKay, F., and J.L. Browning. 1998. Turning off follicular dendritic cells. Nature. 395: 26-27 [Medline]. |
34. |
Kato, J.,
N. Motoyama,
I. Taniuchi,
H. Takeshita,
M. Toyoda,
K. Masuda, and
T. Watanabe.
1998.
Affinity maturation in Lyn kinase-deficient mice with defective germinal
center formation.
J. Immunol.
160:
4788-4795
|
35. | Xu, J., T.M. Foy, J.D. Laman, E.A. Elliott, J.J. Dunn, T.J. Waldschmidt, J. Elsemore, R.J. Noelle, and R.A. Flavell. 1994. Mice deficient for the CD40 ligand. Immunity. 1: 423-431 [Medline]. |
36. | Kelly, B.S., J.G. Levy, and L. Sikora. 1979. The use of enzyme-linked immunosorbent assay (ELISA) for the detection and quantification of specific antibody from cell cultures. Immunology. 37: 45-52 [Medline]. |
37. | Rose, M.L., M.S.C. Birbeck, V.J. Wallis, J.A. Forrester, and A.J.S. Davies. 1980. Peanut lectin binding properties of germinal centers of mouse lymphoid tissue. Nature. 284: 364-366 [Medline]. |
38. | Hardy, R.R., K. Hayakawa, D.R. Parks, L.A. Herzenberg, and L.A. Herzenberg. 1984. Murine B cell differentiation lineages. J. Exp. Med. 159: 1169-1188 [Abstract]. |
39. | Lalor, P.A., G.J.V. Nossal, R.D. Sanderson, and M.G. McHeyzer-Williams. 1992. Functional and molecular characterization of single, (4-hydroxy-3-nitrophenyl)acetyl (NP)- specific, IgG1+ B cells from antibody-secreting and memory B cell pathways in C57BL/6 immune response to NP. Eur. J. Immunol. 22: 3001-3011 [Medline]. |
40. | Kimoto, H., H. Nagaoka, Y. Adachi, T. Mizuochi, T. Azuma, T. Yagi, T. Sata, S. Yonehara, Y. Tsunetsugu-Yokota, M. Taniguchi, and T. Takemori. 1997. Accumulation of somatic hypermutation and antigen-driven selection in rapidly-cycling surface Ig+ germinal center (GC) B cells which occupy GC at a high frequency during primary anti-hapten response in mice. Eur. J. Immunol. 27: 268-279 [Medline]. |
41. | Ahearn, J.M., M.B. Fischer, D. Croix, S. Goerg, M. Ma, J. Xia, X. Zhou, R.G. Howard, T.L. Rothstein, and M.C. Carroll. 1996. Disruption of the Cr2 locus results in a reduction in B-1a cells and in an impaired B cell response to T-dependent antigen. Immunity. 4: 251-262 [Medline]. |
42. |
Molina, H.,
V.M. Holers,
B. Li,
Y.-F. Fang,
S. Mariathasan,
J. Goellner,
J. Strauss-Schoenberger,
R.W. Karr, and
D.D. Chaplin.
1996.
Markedly impaired humoral response in mice
deficient in complement receptors 1 and 2.
Proc. Natl. Acad.
Sci. USA.
93:
3357-3361
|
43. |
Maeda, K.,
G.F. Burton,
D.A. Padgett,
D.H. Conrad,
T.F. Huff,
A. Masuda,
A. Szakal, and
J.G. Tew.
1992.
Murine follicular dendritic cells (FDC) and low affinity Fc-receptors for
IgE (Fc![]() |
44. |
Han, S.,
S.R. Dillon,
B. Zheng,
M. Shimoda,
M.S. Schlissel, and
G. Kelsoe.
1997.
V(D)J recombinase activity in a subset
of germinal center B lymphocytes.
Science.
278:
301-305
|
45. |
Takahashi, Y.,
P.R. Dutta,
D.M. Cerasoli, and
G. Kelsoe.
1998.
In situ studies of the primary immune response to
(4-hydroxy-3-nitrophenyl)acetyl. V. Affinity maturation develops in two stages of clonal selection.
J. Exp. Med.
187:
885-895
|
46. | Jacob, J., R. Kassir, and G. Kelsoe. 1991. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl) acetyl. I. The architecture and dynamics of responding cell populations. J. Exp. Med. 173: 1165-1175 [Abstract]. |
47. | Jacob, J., and G. Kelsoe. 1992. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. II. A common clonal origin for periarteriolar lymphoid sheath- associated foci and germinal centers. J. Exp. Med. 176: 679-687 [Abstract]. |
48. | Jacob, J., J. Przylepa, C. Miller, and G. Kelsoe. 1993. In situ studies of the primary immune response to (4-hydroxy-3-nitrophenyl)acetyl. III. The kinetics of V region mutation and selection in germinal center B cells. J. Exp. Med. 178: 1293-1307 [Abstract]. |
49. | Jack, R.S., T. Imanishi-Kari, and K. Rajewsky. 1977. Idiotypic analysis of the response of C57BL/6 mice to the (4-hydroxy-3-nitrophenyl)acetyl group. Eur. J. Immunol. 8: 559-565 . |
50. | Makela, O., and K. Karjalainen. 1977. Inherited immunoglobulin idiotypes of the mouse. Immunol. Rev. 34: 119-138 [Medline]. |
51. | Reth, M., T. Imanishi-Kari, and K. Rajewsky. 1979. Analysis of the repertoire of anti-NP antibodies in C57BL/6 mice by cell fusion. II. Characterization of idiotopes by monoclonal anti-idiotope antibodies. Eur. J. Immunol. 9: 1004-1013 [Medline]. |
52. | Cumano, A., and K. Rajewsky. 1985. Structure of primary anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies in normal and idiotypically suppressed C57BL/6 mice. Eur. J. Immunol. 15: 512-520 [Medline]. |
53. |
Matsumoto, M.,
Y.-X. Fu,
H. Molina,
G. Huang,
J. Kim,
D.A. Thomas,
M.H. Nahm, and
D.D. Chaplin.
1997.
Distinct
roles of lymphotoxin ![]() |
54. | Maeda, K., M. Matsuda, and Y. Imai. 1995. Follicular dendritic cells: structure as related to function. Curr. Top. Microbiol. Immunol. 201: 119-139 [Medline]. |
55. |
Kapasi, Z.F.,
D. Qin,
W.G. Kerr,
M.H. Kosco-Vilbois,
L.D. Shultz,
J.G. Tew, and
A.K. Szakal.
1998.
Follicular dendritic
cell (FDC) precursors in primary lymphoid tissues.
J. Immunol.
160:
1078-1084
|
56. | Kosco-Vilbois, M.H., H. Zentgraf, J. Gerdes, and J.-Y. Bonnefoy. 1997. To `B' or not to `B' a germinal center? Immunol. Today. 18: 225-230 [Medline]. |
57. | Weiss, U., R. Zoebelin, and K. Rajewsky. 1992. Accumulation of somatic mutants in the B cell compartment after primary immunization with a T cell-dependent antigen. Eur. J. Immunol. 22: 511-517 [Medline]. |
58. | McHeyzer-Williams, M.G., M.J. McLean, P.A. Lalor, and G.J.V. Nossal. 1993. Antigen-driven B cell differentiation in vivo. J. Exp. Med. 178: 295-307 [Abstract]. |
59. | Casson, L.P., and T. Manser. 1995. Random mutagenesis of two complementarity determining region amino acids yields an unexpectedly high frequency of antibodies with increased affinity for both cognate antigen and autoantigen. J. Exp. Med. 182: 743-750 [Abstract]. |
60. |
Bothwell, A.L.M.,
M. Paskind,
M. Reth,
T. Imanishi-Kari,
K. Rajewsky, and
D. Baltimore.
1981.
Heavy chain variable
region contribution to the NPb family of antibodies: somatic
mutation evident in a ![]() |
61. | Allen, D., T. Simon, F. Sablitzky, K. Rajewsky, and A. Cumano. 1988. Antibody engineering for the analysis of affinity maturation of an anti-hapten response. EMBO (Eur. Mol. Biol. Organ.) J. 7: 1995-2000 [Abstract]. |
62. | Gray, D., and T. Leanderson. 1990. Expansion, selection and maintenance of memory B-cell clones. Curr. Top. Microbiol. Immunol. 159: 1-17 [Medline]. |
63. | MacLennan, I.C.M., Y.J. Liu, S. Oldfield, J. Zhang, and P.J.L. Lane. 1990. The evolution of B-cell clones. Curr. Top. Microbiol. Immunol. 159: 37-63 [Medline]. |
64. | Liu, Y.-J., J. Zhang, P.J.L. Lane, E.Y.-T. Chan, and I.C.M. MacLennan. 1991. Sites of specific B cell activation in primary and secondary responses to T cell-dependent and T cell-independent antigens. Eur. J. Immunol. 21: 2951-2962 [Medline]. |
65. | MacLennan, I.C.M., A. Gulbranson-Judge, K.-M. Toellner, M. Casamayor-Palleja, E. Chan, D.M.-Y. Sze, S.A. Luther, and H.A. Orbea. 1997. The changing preference of T and B cells for partners as T-dependent antibody responses develop. Immunol. Rev. 156: 53-66 [Medline]. |
66. |
Liu, Y.-J..
1997.
Sites of B lymphocyte selection, activation,
and tolerance in spleen.
J. Exp. Med.
186:
625-629
|