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From Schering-Plough, Laboratory for Immunological Research, 69571 Dardilly, France
The immune system appears to be rigid, restricting one
lymphocyte to make one antibody (1) and the peripheral B cell pool to a constant number (108 in mice) (2-
4). To make a rapid immune response to an unlimited number of antigens at any anatomical site, it has developed
at least three major strategies: (a) continuous production of
2 × 107 lymphocytes/d from bone marrow (mice; 5), displaying a part of the 1011 potential immunoglobulin repertoire (6); (b) the establishment of a long-lived B cell pool
(108), under the influence of environmental antigens (7)
or an idiotypic network (8, 9); and (c) the ability of long-lived B cells to migrate between different lymphoid tissues
(10, 11), thus monitoring sites of antigen invasion. Since
the 108 peripheral B cells have an average life span of
months, <10% of the 2 × 107 B cells produced every day are
expected to be recruited into the long-lived peripheral B cell
pool (2). The question is: where and how does this selection take place?
Most newly produced B cells
from bone marrow first migrate into the spleen through the
terminal branches of central arterioles, arriving in the marginal zone blood sinusoids (Fig. 1 A). Both newly produced
B cells and long-lived recirculating B cells can penetrate
the marginal zone sinus, reaching the outer zone of the periarteriolar lymphocytic sheath (outer periarteriolar lymphoid sheath [PALS]). Although the long-lived recirculating B cells migrate further into the follicle, the majority of
newly produced B cells appear to die within the outer PALS
(2, 12). Thus, outer PALS may represent the site where a
small proportion of the newly produced B cells are recruited into the long-lived pool. The selection signal has
the following features: (a) it selects B cells according to heavy
chain of the valuable region of Ig gene (IgVH) expression
(7); (b) it may respond to environmental antigen at a low
dose (7) or an idiotypic element such as serum Ig (8); (c) it
appears to positively select cells (7); and (d) it does not induce somatic hypermutation and isotype switch (7, 13).
Three aspects of this ligand selection process are unclear: (a)
Does the ligand induce B cell activation and proliferation? (b) How does this ligand selection differ from the negative
selection of autoreactive B cells in the periphery? and (c)
What is the mechanism in follicles that makes B cells become long lived?
Recently, the molecular mechanism underlying B cell
migration from the outer PALS towards follicles was partially revealed by a series of mice lacking TNF- The development of immunohistological techniques for detecting antigen-specific plasma cells (19) and antigen-specific B cells (20) have permitted an analysis of the microenvironments for antigen-specific B cell responses. A common
picture appearing early in spleens during immune responses
to all types of antigens is the accumulation of antigen-specific proliferating B cells within the outer PALS (Fig. 2;
19-23). In response to T cell-dependent antigen (TD; protein), the proliferating B cells within the outer PALS either
migrate into the follicles to initiate the germinal center reaction, or undergo plasma cell differentiation within the
PALS (Fig. 2, A and B). In response to T cell-independent type 1 antigen (TI-1; lipopolysaccharide), even though there
is an impressive plasma cell reaction within the outer PALS
and red pulp, follicular B cell proliferation is moderate (Fig.
2 C). In response to TI-2 (polysaccharide), most proliferating B cells differentiate into plasma cells (Fig. 2 D). In secondary TD responses, there is a robust B cell proliferation
and plasma cell differentiation within the outer PALS.
However, the follicular B cell proliferation is less extensive
compared to that of primary responses (22, 24).
Double transgenic mice coexpressing self-antigen and anti-self-antibody have revolutionized our understanding of the mechanisms underlying immunological tolerance (25, 26). A striking phenomenon observed by Cyster
and co-workers was that within the splenic outer PALS,
self-reactive B cells were stopped from migrating into the B
cell follicles and they died within the outer PALS (27, 28).
This antigen receptor-mediated inability of follicular homing was suggested to be most closely linked to the failure of
self-reactive B cells to compete with other B cells for the follicular niches in the presence of self-antigen (Fig. 1, B
and C). A similar phenomenon was observed by Fulcher
and Basten (29). However, they suggested that the inability
of follicular homing was caused by a strong antigen receptor engagment, which was independent of other B cells
(29). These different views might be due to the different
interpretations of available self-antigen (hen egg lysozyme;
HEL) concentration in double transgenic mice expressing both anti-HEL antibody and HEL antigen and in single
transgenic mice expressing only HEL antigen. Fulcher and
Basten estimated that although single transgenic mice contained 15 ng/ml sera HEL antigen, double transgenic mice
contained 9 ng/ml sera HEL antigen, possibly due to the
fixation of HEL antigen by anti-HEL antibodies on B cells
and in secreted form. Accordingly, when anti-HEL B cells
were transferred into double transgenic mice, the 9 ng/ml sera HEL presumably triggered only 26% of the antigen receptors on B cells, which may be under the threshold
needed to arrest B cells within the outer PALS and to prevent their entry into the follicles (Fig. 1 B). In contrast, the
15 ng/ml sera HEL antigen in single transgenic mice presumably triggered 47% of the antigen receptors on B cells,
which may be well above the threshold (Fig. 1 C). This interpretation was questioned by the accuracy of serum antigen measurement (30) and by the uncertainty that this relatively small difference in degree of receptor engagement
(26 versus 47%) could exert such a major difference in the
capacity of cell homing to follicles (31). To clarify this issue, it will be necessary to measure the intracellular biochemical signals within HEL-specific B cells after incubation with sera from double and single transgenic mice,
respectively.
In this issue of The Journal of Experimental Medicine, Cook
et al. report a series of experiments analyzing several factors that may control the follicular homing of B cells (32): (a) dose and duration of antigen triggering; (b) the naive versus tolerant state of anti-HEL B cells; (c) the percentage of
anti-HEL B cells relative to the total B cell population; and
(d) the nature of the competing follicular B cells within the
recipient mice, being either monoclonal syngenec naive B
cells, polyclonal naive B cells, or monoclonal HEL-specific
tolerated B cells. The results allow them to conclude that
(a) B cells undergo arrest in the outer PALS in response to
ligation of a critical number of BCRs; (b) this phenomenon
is not related to the state of B cells, being either naive or
tolerant; and (c) the results appear to be independent of the
composition of follicular B cells within the recipient mice.
In summary, outer PALS arrest seems to be an intrinsic
property of all types of B cells in response to BCR triggering by all types of antigens. These together with the facts
that (a) both normal B cells and tolerant B cells require a
similar dose of antigen to be arrested within the outer PALS
and (b) tolerant B cells rapidly increase in their size after
transfer into HEL transgenic mice (29) suggest that the tolerant B cells undergo antigen-driven abortive activation within
the outer PALS, which prevent their further migration into
the follicle.
An important conclusion derived from the elegant studies using antigen and antigen receptor transgenic mice is
that the accumulation of antigen-specific B cells within the
outer PALS in the first few days of immune response is
critical for their encounter with rare antigen-specific T cells
(for review see reference 30). However, the fate of B cells
arrested in the outer PALS depends on not only T cell help,
but also the types of immunizing antigen and the state of B
cell differentiation and tolerance. In the absence of T cell
help, B cells die in a TD response, but they differentiate
into plasma cells within the outer PALS in a TI-2 response
(19). This may be explained by a TI-2 antigen (a) having reiterative epitopes that strongly cross-link antigen receptors; (b) being presented by specialized cells such as marginal zone macrophages; (c) triggering special B cells such as
B1 cells or marginal zone B cells (33). In a TD response,
the naive B cells and tolerant B cells that accumulate in the
outer PALS behave differently with respect to helper T
cells. Although helper T cells allow naive B cells entering
the follicle to initiate the germinal center reaction or to differentiate into plasma cells within the outer PALS, these T
cells kill tolerant B cells upon encounters (28, 32, 34). This
is because the Fas ligand possibly expressed by these helper
T cells kills B cells when their antigen receptors are either
not engaged or do not have a normal intracellular signal
transduction pathway (35). Naive B cells and memory
B cells that have accumulated in the outer PALS also respond differently to helper T cells in TD responses. Although naive B cells preferentially migrate into the follicle to initiate the GC reaction, memory B cells preferentially
undergo terminal plasma cell differentiation within the
outer PALS (21).
In conclusion, the splenic outer PALS represents a critical site where B cells undergo antigen-driven selection, activation, and deletion. It will be important to further analyze the cellular composition and cellular trafficking in this
important anatomical site. For example, it appears that the
outer PALS has reduced numbers of DCs, the key antigen-presenting cells in the initiation of primary TD responses
(38). But a study by De Smedt et al. showed that a population of marginal zone DCs rapidly migrated into the outer
PALS after administration of LPS into mice (39). Thus, the
outer PALS represents a site where antigen-specific T and
B cells as well as DCs meet after immunization. The recent discovery of chemokine receptors as HIV coreceptors has
boosted the discovery of large numbers of chemokines and
chemokine receptors by genomic programs. A collection of
mice lacking these molecules will be expected to be generated during the next few years. These mice, together with
those lacking TNF members/TNF receptors (for reviews
see references 40) will help us to further understand the
mechanisms of cellular migration and interaction within
secondary lymphoid tissues during morphogenesis, immune response, and immune tolerance.
Fig. 1.
The structure of splenic
white pulp and the migration pathways of B lymphocytes. (A) Double
anti-IgM (red) and anti-IgD (blue) staining of a splenic section of a nonimmunized rat. Although nonrecirculating IgM+IgD marginal zone B cells
are stained in red, recirculating
IgM+IgD++ B cells are stained in blue.
The black circles show the proposed migration pathway of a recirculating B
lymphocyte. It enters the spleen marginal zone (MZ) through a terminal
branch of a central arteriole. It crosses
the marginal zone sinus and migrates
into a primary follicle. After some
hours, it leaves the follicle, migrates
along the outer PALS, and finally
reaches the red pulp through a bridging
channel. (B) The migration pathway of
HEL-specific B cells after transfer into
HEL and anti-HEL double transgenic mice. (C) The migration pathway of
HEL-specific B cells after transfer into
HEL single transgenic mice (27).
[View Larger Version of this Image (86K GIF file)]
(14), lymphotoxin
(15), lymphotoxin
(16), TNFR1 (15), and a
putative chemokine receptor Burkitt's lymphoma receptor
1 (BLR1) (17). In all these mice, IgD+ B cells were retained within the outer PALS. Importantly, BLR1 was
shown to be selectively expressed on mature recirculating B
cells, but not on newly produced B cells. This suggests that BLR1 is the navigator that directs the migration of B cells
from the outer PALS to the follicle. It will be interesting to
know (a) if the regulation of BLR1 expression is by TNF
family members or by antigen receptor complex triggering;
and (b) if follicular dendritic cells or germinal center dendritic cells (DCs; 18) produce the BLR1 ligand(s).
Fig. 2.
Outer PALS arrest
of anti-DNP-specific B cells in
T cell-dependent and -independent antibody responses. (A) 48 h
after immunization with DNP
protein antigen in the rat, antigen-specific proliferating plasma
blasts and B blasts can be respectively observed within the outer
PALS and follicle of spleen. Blue
stains DNP-binding cells; red
stains BrdU; brown stains T cells.
(B) Schematic representation of
A. (C) Schematic representation
of splenic B cell response to TI-1
antigen (DNP-LPS). It shows a
very impressive antigen-specific B cell proliferation and differentiation within the outer PALS and red pulp. In contrast, follicular B cell proliferation is moderate. (D) Schematic representation of splenic B cell response to
TI-2 antigen (DNP-Ficoll). It
shows that most proliferating B
cells within the outer PALS differentiate into plasma cells.
[View Larger Version of this Image (139K GIF file)]
Address correspondence to Yong-Jun Liu, Schering-Plough, Laboratory for Immunological Research, 27 Chemin des Peupliers, BP 11, 69571 Dardilly, France. Phone: 33-4 72-17-27-00; FAX: 33-04-78-35-47-50.
Received for publication 26 June 1997 and in revised form 16 July 1997.
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Lymphotoxin-![]() |