|

Received: 31 October 2018    Accepted: 28 November 2018

DOI: 10.1111/imr.12731

INVITED REVIEW

Germinal center B cell initiation, GC maturation, and the

coevolution of its stromal cell niches

Ann M. Haberman1,2  | David G. Gonzalez1,3 | Patrick Wong1 | 

Ting-ting Zhang1 | Steven M. Kerfoot4

1
Department of Immunobiology, Yale
University, New Haven, Connecticut Summary
2
Department of Laboratory Medicine, Yale Throughout the developing GC response, B cell survival and fate choices made at the
University, New Haven, Connecticut
single cell level are dependent on signals received largely through interactions with
3
Department of Genetics, Yale University,
other cells, often with cognate T cells. The type of signals that a given B cell can en-
New Haven, Connecticut
4
Department of Microbiology and counter is dictated by its location within tissue microarchitecture. The focus of this
Immunology, Western University, London, review is on the initiation and evolution of the GC response at the earliest time
ON, Canada
points. Here, we review the key factors influencing the progression of GC B cell
Correspondence ­differentiation that are both stage and context dependent. Finally, we describe the
Ann M. Haberman, Department of
Immunobiology, Yale University, New Haven, coevolution of niches within and surrounding the GC that influence the outcome of
Connecticut. the GC response.
Email: ann.haberman@yale.edu

KEYWORDS
Funding information
Multiple Sclerosis Society of Canada; B cells, cell differentiation, cytokines, lineage commitment/specification, stromal cells,
National Institute of Arthritis and transcription factors
Musculoskeletal and Skin Diseases, Grant/
Award Number: P30AR053495; National
Institute of Allergy and Infectious Diseases,
Grant/Award Number: R01AI080850 and
R21AI101704

1 | I NTRO D U C TI O N
Effective immune responses to pathogens and vaccines critically
depend on the generation of high-­affinity antibodies. The steady in-
crease in antibody affinity during adaptive immune responses, known
as affinity maturation, requires the formation of an organized cluster of
proliferating B and T lymphocytes in germinal centers (GC). Germinal
center responses are initiated within secondary lymphoid tissues
that are inherently organized, where antigen-­inexperienced T and B
cells are separated into T-­cell zones and B cell follicles. CD4+ T cells
expressing T-­cell receptors (TCRs) specific for newly arriving antigen
are activated to both proliferate and differentiate through interactions
with dendritic cells (DCs) that have internalized antigen and presented
proteolytically degraded fragments on surface MHC II molecules. At
This article is part of a series of reviews covering Germinal Center and Extrafollicular
Immune Responses appearing in Volume 288 of Immunological Reviews.
the same time, antigen-­specific B cells become activated after encoun-
tering antigen in its intact, unprocessed form and experiencing B c­ ell
receptor (BCR) ligation. B cells that have internalized antigen bound to
its specific BCR can then also present processed components in the
context of surface MHC II. A series of coordinate interactions between
activated T and B cells specific for the same (cognate) antigen are the
foundation for B cell activation throughout the GC response, both
during the initial pre-­GC phase and within the mature GC.
The GCs that emerge within B cell follicles of secondary lym-
phoid tissue are highly organized structures, within which GC B cells
compete to acquire additional antigen and engage follicular helper
T cells. With contemporary thinking, GC B cells expressing BCR
with higher affinities are selected to further proliferate, while less
fit B cells expressing BCRs with weaker affinities may not receive
sufficient survival or instructive signals and undergo apoptotic cell
death. In addition to survival, T/B interactions also drive differenti-
ation of GC B cells into one of two lineages that are fundamental to

10  |  wileyonlinelibrary.com/journal/imr
© 2019 John Wiley & Sons A/S. Immunological Reviews. 2019;288:10–27.
Published by John Wiley & Sons Ltd
HABERMAN et al.
durable immunity; long-­lived high-­affinity plasma cells and memory
B cells.1-4 In this way, GCs mold, amplify, and add persistence to the
most effective B cells of the immune response.
Throughout the developing GC response, B cell survival and fate
choices made at the single cell level are dependent on signals received
largely through interactions with other cells, often with cognate T
cells. The type of signals that a given B cell can encounter is dictated
by its location within tissue microarchitecture. A major component
of B cell activation and differentiation in the GC response involves
sequential changes in migration patterns that move the cell from one
specialized zone to the next. The focus of this review is on the initia-
tion and evolution of the GC response at the earliest time points. We'll
review the key factors influencing the progression of GC B cell differ-
entiation that are both stage and context dependent. The review will
finish with a discussion of the niches within and surrounding the GC
that may influence the outcome of the GC response.
2 | L A N D M A R K S A N D U N I Q U E FE AT U R E S
O F E S TA B LI S H E D G C S
2.1 | Tissue compartments of mature GCs
To better appreciate the similarities and disparities of the initial GC
response, we begin with an overview of the fundamentals of fully
mature GCs. As the newly established GC expands, non-­responding
B cells are peripherally displaced and surround the emerging GC,
forming what is known as the follicular mantle. Mature GCs have
a complex architecture that is comprised of two prominent subdo-
mains, the light zone (LZ) and dark zone (DZ), originally named for
their relative appearance in hematoxylin/eosin-­stained tissue sec-
tions.5-9 The LZ is distinguished by the presence of the fine pro-
cesses of a stromal cell subset, follicular dendritic cells (FDCs), which
entirely comprise the stromal cell matrix in this region and form a
8
reticular network that GC B cells infiltrate. Within primary follicles
FDCs are a key source of CXCL13, a chemokine that is requisite for
the correct organization of GCs into compartmentalized DZ and
10
LZs. FDCs retain deposits of activated complement components
and antigen-­antibody complexes via the low-­affinity Fc receptor for
IgG (FcγRIIb) and the complement receptors CD21 and CD35.11-15
FDCs can serve as long-­term depots of undegraded Ag,11,13,15-17 and
for this reason they have classically been considered an important
site of antigen presentation to B cells. Intrafollicular CD4+ T-­helper
cells known as T follicular helpers (Tfh) are predominantly found
within the LZ, sometimes in great numbers, but are also present
5,18,19
throughout the follicle mantle. Tfh are critical to the formation
and maintenance of a productive GC, largely due to their provision
20-26
of IL-­21, IL-­4, and CD40. At the peak of the response, Tfh can be
distinguished from other cell subsets by a high level of expression of
the molecules PD-­1, ICOS, and the chemokine receptor CXCR5. 27-30
The DZ contains a higher density of proliferating cells and har-
bors another stromal cell type that produces CXCL12, the ligand
for CXCR4, and lacks the distinctive CD35 expression of FDCs.31,32
The majority of DZ-­resident B cells, also referred to as centroblasts,
|
      11
GC
Follicle
Interfollicular zone
Sub-SCS
T/B border
T cell zone
FDC
F I G U R E   1   Lymph node tissue compartments influencing the
developmental stages of germinal center B cells
express lower levels of a variety of surface markers, including BCRs,
CD40, and CD86, but a higher level of Bcl6 and CXCR4 compared
to their LZ counterparts.32-34 Many LZ-­resident GC B cells, aka cen-
trocytes, are smaller in size and are typically CD86hi, CD83hi, and
CXCR4lo.33 Although these phenotypic differences have come to be
viewed as surrogate markers of GC B cell location, it is important
to note that B cells residing within each zone are far from homoge-
neous. GCs are consistently oriented within B cell follicles (Figure 1).
The LZ is reproducibly more proximal to the subcapsular sinus of
lymph nodes or the marginal sinus enveloping the white pulp of
spleens. The DZ balloons beneath the FDC network, extending to-
ward the border of the follicle with the T-­cell zone.
2.2 | Selection of higher-­affinity variants, interzonal
migration, and cyclic reprogramming
During their extensive clonal expansion, B cells with higher-­affinity
BCR variants are selectively enriched.35-38 GC B cells express high
levels of activation induced cytidine deaminase that drives a unique
process of somatic hypermutation (SHM) of immunoglobulin (Ig)
genes that can introduce amino acid substitutions in the antibodies
produced.39,40 Because SHM is introduced during DNA replication, it
was thought to occur in the DZ where mitotic cells are more evident,
especially in tonsils.32,33,41,42 Selection for higher-­affinity B cells has
been proposed to result from competition for the acquisition of anti-
gen.6,43,44 As FDCs are the most evident site of antigen accumulation
within follicles, and have the ability to promote the survival of GC
B cells that are prone to apoptosis, the selection process is thought
to occur within the LZ compartment.45-47 Early thinking on GC B ­cell
selection envisioned that a key element involved the crosslinking
of BCRs while in contact with antigen-­laden FDCs.48-50 However,
a recent study by Luo et al51,52 indicates that some aspects of ca-
nonical BCR signaling are rewired in Bcl6hi GC B cells, such that
BCR crosslinking per se cannot fully instruct the extensive self-­
renewal observed within mature GCs. Other studies strongly sup-
port a model in which positive selection requires GC B cells to form
immunological synapses with Tfh cells53-55 and reviewed in.56,57 B
cells with higher affinity acquire more protein antigen, resulting in
increased surface presentation of MHC II-­antigen complexes, and
a greater capacity to stimulate Tfh cells with a cognate specific-
ity.33,58-62 It is important to note that BCR signaling during antigen
 |
12       HABERMAN et al.
acquisition and Tfh cell engagement need not be mutually exclusive
and could be synergistic. In support of this, the combination of BCR
and CD40 agonism more robustly invokes cMYC expression and fur-
ther cell division than either alone.52 Other models for affinity matu-
ration postulated forms of negative selection, which could operate
48,63-66
in concert with positive selection.
To reconcile a perceived need for sequential events to occur in
spatially separated regions of the GC, the “cyclic re-­entry” model
was proposed.67,68 With a contemporary view of this model, DZ-­
resident B cells that have exited cell cycle lose the transcriptional
profile typical of centroblasts and with lower CXCR4 levels subse-
quently migrate to the LZ.10,32,33,69 Intravital imaging studies of GC
B cell migration patterns are consistent with infrequent movement
59,70-72
between compartments. Within the LZ, the recent immigrants
compete for access to antigen, potentially experience BCR crosslink-
ing during antigen acquisition, and then compete for contacts with
Tfh cells that selectively provide factors such as CD40L, IL-­21, and
52,73,74
IL-­4. Foxo1, cMyc, and AP4 are among the transcription fac-
tors known to play a critical role in the transition of such positively
selected centrocytes to the centroblast transcriptional program, al-
though expression of these transcription factors (TFs) can be short
34,75-78
lived. Thus, the LZ is currently seen as the compartment in
which both self-­renewal and differentiation toward long-­lived effec-
tor lineages are initiated, the latter evidenced by the expression of
antibody secreting cell (ASC)-­associated TFs by B cells within the LZ
55,79-82
harboring the majority of Tfh cells. Self-­renewing GC B cells
undergo a centrocyte to centroblast transition that is characterized
by a reciprocal increase in CXCR4, a decrease in costimulatory mol-
ecules, initiation of cell division, and their return to the DZ to com-
33
plete cell division there. Within the DZ, centroblasts reduce their
surface BCR and recycle existing MHC II complexes, and in this way
have an opportunity to better express their newly mutated BCR that
can be more fully tested when antigen is next encountered.83,84
The bulk of research on GCs has interrogated mature GCs.
However, recent studies have implications on its initiation and de-
velopment. The focus of the remainder of this review is on the pro-
gression of the GC response at the earliest time points, both of the
responding B cells and within the evolving stromal compartments
that coordinate the context of their cellular interactions.
3 |  T- ­B C E LL CO NTAC T S D U R I N G G C
I N ITI ATI O N
Exploration of the earliest stages of GC development had long been
hampered by the seemingly inscrutable developmental steps that
preceded the appearance of the classical surface markers of GC B
cells. Markers typically ascribed to GC B cells include high levels of
CD95 (Fas), the GL7 epitope, a loss of IgD, and an increased ability to
85-87
bind peanut agglutinin (PNA). B cells within established murine
GCs have uniformly low levels of CD38 in mice, while curiously ex-
88
actly the opposite pattern is seen with human GC B cells. However,
it is important to note that during the early phases of an immune
response, some of these markers are also expressed by developing
plasmablasts.89,90 A further challenge to the study of early B cell ac-
tivation events is the downregulation of surface BCR after crosslink-
ing, making antigen specificity hard to confirm in some cases. A
superior indicator of this lineage commitment emerged when the
requisite role of Bcl6 was demonstrated, empowering investigations
into the early steps in this pathway that were previously enigmatic.
Expression of the transcriptional repressor Bcl6 is required for both
Tfh and GC B cell differentiation, a process that involves shifts in
the transcription of other influential TFs.91-95 In order to more easily
identify and track the behavior of antigen-­specific T and B cells, we
and others have developed strains of mice that ubiquitously express
fluorescent proteins such as GFP or RFP and harbor transgenic or
knockin BCR or TCR alleles. With the transfer of such fluorescently
tagged cells, specific cells can be interrogated while remaining ag-
nostic on all other phenotypic aspects during their activation and as
they undergo significant change during subsequent differentiation.
The creation of a productive GC involves step-­wise changes to
the transcriptional profiles and phenotype of responding lympho-
cytes, often including shifts in their chemotactic tendencies. Prior
to activation, antigen-­naïve B and T cells are segregated into B cell
follicles and T-­cell zones (Figure 1). This is the result of migration
patterns not dictated by structural or anatomical limits, but rather
by localized expression of chemotactic agents drawing in B and
T cells to their respective zones based on their expression of recep-
tors for those agents. By virtue of B cell expression of CXCR5, they
are attracted to its ligand chemokine CXCL13 that is produced by
FDCs centrally positioned in follicles96,97 and by marginal reticular
cells (MRCs) that line the subcaspular sinus (SCS) of LNs and splenic
marginal sinus (SMS).98-100 T-­cell localization to the T-­cell zone is me-
diated by their expression of CCR7, the receptor for CCL19/21.101,102
An additional chemoattractant within lymphoid tissue includes the
compound EBI2L that is enzymatically modified by stroma lining the
SCS and SMS. EBI2L is also found at the cortical sinuses descending
through the interfollicular (IF) regions, at the border between T-­cell
zones and follicles (T-­B border), as well as at the bridging channels
positioned between follicles in spleens (Figure 1).103-109 The com-
binatorial expression of even just these three chemoattractants by
stromal cell populations establishes multiple distinct architectural
domains within which lymphocytes with similar tropisms can aggre-
gate (Figure 1).
With an influx of inflammatory material via lymph to lymph nodes,
or via blood in the spleen, B and T cells specific for any newly arriving
antigens become activated and adjust their migratory propensities.
Within lymph nodes, naïve B cells within follicles first encounter
their cognate antigen delivered via lymphatic vessels, cortical si-
nuses, or at the surface of SCS lining macrophages.110-116 Exposure
to pathogen-­associated molecular motifs and BCR crosslinking result
in increased levels of CCR7, CD86, and MHC II, as well as receptivity
to CD40 signaling.117,118 With this increase in CCR7, activated B cells
rapidly relocate to the periphery of the follicle119-121 and are retained
in part by further expression of EBI2.103,104 Activated CD4+ T cells
also adjust their chemotactic propensities by an increase in their
HABERMAN et al.
expression of CXCR5 and EBI2.96,122 As a result, recently activated
+ + + +
CD4 T cells and B cells are similarly CXCR5 , CCR7 , and EBI2 and
both migrate to and congregate at the T-­B border and interfollic-
ular regions within LNs or the analogous bridging channels within
spleens. After engaging T cells that share their cognate specificity,
activated B cells commit to one of several potential fates; they either
re-­enter the follicle and commit to the formation of a new GC, or
they differentiate outside of GCs into low-­affinity short-­lived ASCs,
123-125
early memory B cells, or other effector B cell lineages. It is at
these locations that elevated Bcl6 protein levels are first apparent
in both responding B and T cells.126,127 Once Bcl6-­mediated tran-
scriptional repression is fully enforced in nascent GC B cells and Tfh
cells, they lose expression of chemokine receptors such as EBI2 and
CCR7 that retained them at the follicle periphery, resulting in revised
chemotactic patterns that instead drive them to the follicle interior
shortly thereafter.108,127-130
Importantly, the requirements for commitment to the intrafollic-
ular GC pathway differ for T and B cells. It is clear that T-­cell differ-
entiation to the perifollicular Tfh state can be driven initially by their
interactions with DCs.131 Bcl6 expression by a subset of responding
T cells is detectable very early, within 1-­2 days of antigen encoun-
126,132,133
ter. Interestingly, typical Tfh markers CXCR5 and PD-­1 are
quickly upregulated on many responding T cells, but after 3 days of
interactions with B cells at the follicle periphery are then only main-
tained on a population that ultimately gains entry to the follicle and
continues to interact with B cells.126,132-135 In contrast, full activation
of B cells to commit to the GC pathway depends on those B cells
interacting with cognate T cells.
Over the course of several days, cognate B and T cells form sta-
ble immunological synapses, but repeatedly engage new partners
during this protracted and formative time period, all the while re-
maining at the follicle periphery. Bcl6 expression by B cells is not
detectable until after the emergence of Bcl6+ Th cells. Thus, B cell
commitment to the GC pathway, re-­entry to the follicle, and the co-
alescence to form a proliferative foci there requires several days of
ongoing serial interactions with perifollicular Tfh. Interestingly, Tfh
126
entry into the follicle proceeds that of GC B cells, suggesting that
the availability of Tfh support within the follicle is a prerequisite for
the survival of newly arriving GC B cells. Early, pre-­GC cognate in-
teractions that occur at the follicle periphery are therefore critical to
establishing the GC and determining immune outcome.
4 | PRO G R E S S I V E G C B C E LL
D I FFE R E NTI ATI O N H A S D I S TI N C TLY
R EG U L ATE D S TAG E S
4.1 | Germinal center B cells are developmentally
delayed
Shortly after BCR signaling in vitro and in vivo, activated B cells
are well poised to engage Th cells and respond to CD40 liga-
tion.117 However, many cell divisions are required before Bcl6
26
protein expression becomes evident in responding B cells. This
|
      13
is true even under conditions in which activation and cell con-
tacts are occurring with rapidity. The delay in the appearance
of GC B cells had been a long-­s tanding paradox. Multiple mod-
els have proposed mechanisms that may influence the differen-
tiation of activated B cells to either the GC or ASC lineages. In
vitro studies have suggested that B cell ­f ate decisions could be
stochastic and B cell intrinsic.136-138 Alternatively, the initial BCR
signal strength139,140 or competition for antigen and its presen-
tation to cognate Th cells141 may be deciding factors. However,
the delay in GC B cell development could also be influenced by
other potential constraints. For example, Tfh cells require mul-
tiple days to complete their effector differentiation, but once
complete might be capable of instructing GC B cell differentia-
tion expeditiously. A teleologic explanation for the delay in GC B
cell formation might instead relate to the need for multiple line-
age fates to be created from every antigen-­s pecific B cell after
its initial activation. Perhaps multiple rounds of proliferation are
key to ensuring that an investment in the GC response only oc-
curs after the production of early memory and short-­t erm ASC B
cells. Here, we review the results of recent studies that suggest
additional mechanisms and provide some insights and into these
aspects of B cell differentiation.
4.2 | Impact of CD40 signaling
B ­cell lineage commitment is governed by opposing transcriptional
networks that can be mutually antagonistic. Bcl6 acts as a transcrip-
tional repressor of the TFs IRF4 and BLIMP1, and whereas it is es-
sential for GC B c­ ell formation,93,127,142-144 when overexpressed it
can prevent ASC formation.145-147 Reciprocally, high levels of IRF4
or induction of BLIMP1 represses transcription of Bcl6 but facilitates
ASC differentiation.148,149 To further confound our understand-
ing of transcriptional regulation, a transient and low expression of
IRF4 promotes the transcription of Bcl6 and is required for proper
GC B cell progression,150 despite the fact that it directly binds to
and represses the Bcl6 promoter when at higher levels.92,151 Thus,
IRF4 is required for both ASC and GC development, although ASC-­
associated genes are promoted when IRF4 is at sustained or high
levels of expression.79,150,152-155
Tfh cells express the surface costimulatory molecule CD40L
and supply the cytokines IL-­4 and IL-­21 to GC B cells, all of which
are critical to the formation of fully mature and productive GCs. 20-
26
The combined expression of IL-­4 and IL-­21 is unique to Tfh cells,
however, all Th-­cell populations defined to date are thought to ex-
press CD40L, and very early in an immune response.156,157 Signaling
via CD40 ligation invokes the non-­canonical NF-­kB pathway and
uniquely the nuclear translocation of heterodimeric RelB/p52.158-160
Although CD40 signaling is required for GC B cell development,1,161
their formation can also be antagonized by chronic CD40 signaling
that instead promotes ASC development.151,162-165 ASC formation
during chronic CD40 signaling is in part mediated by IRF4, a TF
whose nuclear translocation is promoted by CD40 signaling.151,166
These seemingly conflicting roles of CD40 pose a conundrum: CD40
 |
14       HABERMAN et al.
and CD40L are required for GC B cell formation, but continued
CD40 signaling discourages it.
Some insight into the paradoxical roles of CD40 was garnered by
our recent work that examined the temporal relationship between
CD40 signaling and TF shifts during GC B cell differentiation.
this study, B cells were first observed to express intermediate levels
of Bcl6 within the interfollicular regions of LNs. Intriguingly, these B
cells also were found at their first emergence to co-­express RelB and
IRF4 at intermediate levels, factors known to be mutually antagonis-
92,151
tic to Bcl6. Consistent with the presence of nuclear RelB and
IRF4, CD40 signaling was required for this very first step in Bcl6 pro-
tein production. At this early stage, Bcl6int GC precursors retained
some plasticity—the infusion of either anti-­CD40 or additional an-
tigen in vivo at this incomplete stage of development could still di-
26
vert them toward Bcl6lo IRF4hi ASCs. However, it is important to
note that Bcl6lo IRF4hi ASC can be seen prior to the detection of
Bcl6int pre-­GC B cells, 26 an observation that is consistent with the
co-­existence of an alternative pathway for extrafollicular ASC for-
mation that is CD40 dependent but does not involve Bcl6.
Although CD40 signaling may establish a transcriptional land-
scape that is initially compatible with intermediate expression levels
of Bcl6, the presence of RelB and the antagonistic TF it promotes,
IRF4, is incompatible with a complete repression of Bcl6 target genes
within pre-­GC B cells. 26 Consistent with this, pre-­GC B cells display
a partial GC B cell phenotype (CD38int, PNAint). 26 Bcl6 is a member
of the BTB-­POZ zinc finger family of TFs that forms a homodimer
167,168
capable of binding the corepressors SMRT, NCOR, and BCOR.
Bcl6 controls GC B cell differentiation by regulating cell cycle genes,
repressing terminal differentiation factors, and suppressing some
91,169
signaling pathways, including some aspects of BCR signaling.
Repressed target genes in dark zone GC B cells in mice include cd38,
irf4, and prdm1 (encoding BLIMP1).92,143,144 Notably, the extent of re-
pression of potential Bcl6 target genes is heavily influenced by core-
91,147,170,171
pressors that are themselves independently regulated.
Therefore, Bcl6 expression per se does not ensure target repression.
Short-lived ASC
signalin D40
C
g
+
Further
IRF4 +
RelB+
IRF4 +
nuclear RelB+
N?
Bcl6 + Bcl6 ++
CD40 signaling
IL-21
CD40 refractory
phase
IRF4int RelB+
FDC Centrocyte
Bcl6int PreGC B cell
It remains unclear whether the ineffective repression of Bcl6 target
genes in GC B cell precursors is the result of subthreshold levels of
Bcl6, or alternatively the absence of other corepressors that would
otherwise enable effective transcriptional repression.170,172,173
26
In Although CD40 signaling is required for this first step in pre-­GC B
cell development, CD40 signaling must cease at the immediately sub-
sequent stage to allow for that incomplete stage of differentiation to
further progress.26 The intermediate differentiative stage of pre-­GC
B cells is characterized by mutually antagonistic TFs (IRF4int Bcl6int)
and is followed shortly thereafter by the appearance of mature GC
B cells that are Bcl6hi with low levels of IRF4 and RelB and a more
classical phenotype (CD38lo PNAhi).26 Interestingly, the shift from the
immature pre-­GC B ­cell state to the more mature Bcl6hi state involves
both transcriptional and post-­transcriptional regulation,26 the latter
a form of regulation that can be mediated by micro-­RNAs.162,174,175
Importantly, disrupting T-­B interactions or inhibiting CD40 signaling
in vivo during this early transitional stage encourages GC B c­ ell mat-
uration. However, when CD40 agonism is prolonged or additional
soluble antigen is introduced, GC precursors are instead expanded
and shunted away from the intrafollicular GC pathway.26,165 Based on
these results, a model of initial GC B cell differentiation has been pro-
posed to be a multistaged process completed by a transient diminution
in the receipt of T-­cell help within its immediate microenvironment
(Figure 2). Here, it is envisioned that GC B cell differentiation is a two-­
stage process involving first a T-­cell contact and CD40-­dependent
phase that generates RelB+ IRF4+ Bcl6int GC precursors and second,
a maturation phase immediately following that leads to the Bcl6hi in-
trafollicular state that is prevented by further CD40 signaling. Thus,
the impact of CD40 signaling may be stage specific in that it promotes
GC B cell precursor formation, but diverts precursor progression once
formed. It is intriguing that the shifting transcriptional profiles of GC
B cell precursors are staged over the course of multiple cell divisions.
More research is required to understand how the sequential introduc-
tion and subsequent removal of TFs is advantageous to the initial for-
mation of GC B cells.
GC lineage
F I G U R E   2   Temporal and context-­
dependent shifts in transcription
factor composition during GC B cell
Centroblast Plasma cell
development
HABERMAN et al.
It remains unclear whether a transient abstinence from CD40L
stimulation plays a similar role in established GCs. The transition
between the classical centrocyte phenotype to that of an idealized
centroblast may involve progressive changes as well. LZ B cells experi-
encing CD40L stimulation during engagement with Tfh cells gain cMyc
expression, which subsequently promotes expression of AP4, a TF that
is maintained within B cells by IL-­21 signaling.78 Subsequent loss of AP4
requires further cell division(s) with the DZ, a process that is needed to
eventually exit cell cycle.78 The potential impact of additional CD40
signaling by DZ-­resident B cells, and whether it could occur in the ab-
sence of IL-­21, remains unclear. As there are far fewer Tfh cells within
the DZ than the contiguous LZ, this compartment offers a relative
dearth of T-­cell–derived CD40L. The sheltered spaces within the DZ
compartment appear to provide other advantages as well to newly
transitioning centroblasts to GC dark zones. A study by Bannard et al32
indicates that GC B cells unable to escape the Tfh-­rich environment of
the LZ have a competitive disadvantage over time, suggesting that in
the long run the cyclic movement between zones ultimately empowers
a selective advantage in a way that is not yet understood. Although
some analogies can be made between the initial development of GC
B cells at the follicle periphery and the cyclic reprogramming of mature
GCs, there are two major differences—(a) newly arriving Bcl6hi centro-
cytes that are seeking antigen and Tfh help are already experiencing
some level of transcriptional repression by Bcl6, and (b) a reservoir of
antigen is accessible within the FDC network. More research is needed
to untangle this complex series of events.
How might pre-­G C B cells transiently avoid CD40 signaling
and resolve their mixed state of mutually antagonistic TFs? One
means by which RelB+ pre-­G C B cells could lose that status is by
an asymmetric cell division (ACD) that results in one daughter
receiving less RelB. Consistent with this, we have observed an
additional cell division of pre-­G C B cells correlates with reduced
RelB levels and a rise in Bcl6. 26 In this scenario, pre-­G C B cells
could undergo an ACD in which Bcl6 is unequally partitioned to
one of the emerging daughters and RelB to the other, resulting in
two distinct cell fates. ACDs involving an unequal distribution of
Bcl6 have been reported among GC B cells, although its role in GC
maintenance has been questioned. 176,177
Alternatively, engage-
ment with perifollicular cognate T cells could be attenuated by a
subthreshold level of antigen presentation. Multiple cell divisions
could lead to a reduced capacity to present antigen, particularly
under conditions when antigen is limiting within that local micro-
environment. This would be consistent with previous reports of
progressively shorter T-­B interaction lengths with non-­infectious
immunogens. 59,126,127 In further support of this, conditions that
instead create persistent pathogen-­d erived antigen promote ex-
trafollicular ASCs and impair GC responses.178 Moreover, antigen
that has been internalized after BCR capping has been reported
to be asymmetrically distributed between daughters after B cell
division, resulting in two daughters who differ in their ability to
179,180
engage cognate T cells. However, once GC B cells have mi-
grated into the follicle interior, they again have access to a rich
source of antigen within the FDC network.
|
      15
4.3 | Role of IL-­21 and IL-­4 in GC B ­cell
maturation and intrafollicular development
Similar to CD40L, the Tfh-­derived cytokines IL-­4 and IL-­21 play dis-
tinct roles at discreet phases of GC B cell development. Although
many Tfh cells found within mature GCs can produce both IL-­4 and
IL-­21, the cytokine expression profiles of Tfh cells can shift during
their development and can differ at distinct locations within lymphoid
tissue.21-23,181,182 Earlier in adaptive immune responses, perifollicu-
lar Tfh predominantly expresses IL-­21 and less IL-­4,23,182 while the
reverse is true within the distal LZ of fully mature GCs where Tfh
cells are more likely to produce IL-­4 than IL-­21.23 CXCR5+ iNK T cells
positioned at follicular boundaries are also capable of providing IL-­21
and/or IL-­4 and promoting the GC lineage development of B cells that
are presenting glycolipid antigens, but the resulting GCs are short-­
lived and unproductive without the added participation of intrafol-
licular Tfh cells responding to protein antigens.183-186 Importantly, the
delivery of IL-­4 and/or IL-­21 by iNK T cells is insufficient for the for-
mation of sustainable GCs when CD40 signaling is absent.185 Other
innate cell types including eosinophils and basophils are also known
to produce IL-­4 and can function as antigen-­presenting cells to Th2
cells,187-190 but they have not been observed within intrafollicular
GCs188 and it remains unclear whether such innate cell types have
the ability to provide IL-­4 in a directed fashion discreetly to antigen-­
specific B cells.
Both IL-­4 and IL-­21 are critical to GC B cell formation. Although
B cells that are unable to signal via IL-­21R or IL-­4Rs can still develop
GCs in vivo, the stunted GCs that do form are significantly reduced in
size.182,191-194 In vitro, both IL-­21 and IL-­4 can enhance the transcrip-
tion and/or translation of Bcl6 in B cells.195,196 Both cytokines are also
known to promote ASC formation.22,197-201 In the case of IL-­21, an in-
ability to signal via its receptor has a direct effect on B cells, resulting
in lower Bcl6 levels among the more limited number of GC B cells that
form.182,191,192
IL-­4R and IL-­21R complexes activate distinct signaling pathways.
Like other cytokine receptors employing the common gamma chain,
both IL-­21R and IL-­4R signal via the Janus kinase molecules JAK1 and
JAK3.202-205 However, these cytokine receptors activate different
signal transducer and activator of transcription (STAT) molecules.
Whereas IL-­21 predominantly activates STAT3 and STAT1, and to a
lesser extent STAT5,206-209, IL-­4, and IL-­13 uniquely and requisitely
activate STAT6210,211.
The results of a recent study we performed suggest that B cell
signaling by these cytokines may influence GC B cells at different
stages.182 These experiments employed the cell transfer of antigen-­
specific B cells that were either deficient in IL-­21R, STAT6, or both in
order to assess the impact of IL-­21 and IL-­4/IL-­13 to B cells intrinsi-
cally. We found that the initial transition from the perifollicular pre-­GC
B cell state to the intrafollicular stage is reduced in IL-­21R−/− B cells,
but unaffected in STAT6−/− B cells, suggesting that IL-­21 has a greater
influence on differentiation at the time of pre-­GC B cell re-­entry to the
follicle interior.182 Importantly, in the absence of both IL-­21 and IL-­4
signaling pathways, Bcl6+ B cells can still form, however, they fail to
 |
16       HABERMAN et al.

establish intrafollicular GCs.182 These findings support the idea that
GC B cell development undergoes a step-­wise progression in which
the initial steps toward this lineage can occur in the absence of IL-­4
and IL-­21, but is dependent upon CD40L. The Bcl6-­expressing B cells
that form without IL-­21R or STAT6 signaling can persist transiently in
vivo, but their expansion is limited, indicating that GC B cell develop-
ment can be arrested at this stage. This could functionally provide a
key checkpoint in this lineage pathway that would allow for the ini-
tial creation of GC precursors, without committing to their furthered
proliferation. In this way, a diverse repertoire could be potentiated
without a concomitant clonal expansion of antigen-­reactive B cells
for which there are not yet cognate Tfh cells capable of sustaining a
mature GC.
The results of this study further suggest that proper GC form
and function within follicles is highly reliant on both cytokines.182
IL-­21 and IL-­4 play non-­redundant roles in the subsequent matura-
tion and self-­renewal of GC B cells within follicles. Smaller numbers
of intrafollicular GC B cells form in the absence of either of these
signaling pathways, but with aberrant phenotypic features.182 When
GCs mature in the absence of either IL-­21 or STAT6 signaling, the
efficiency in the transition between the centroblast and centrocyte
182,197
states is compromised and self-­renewal is reduced. IL-­21R
B cells can form CXCR4hi centroblasts but they overexpress some
features that are more typical of centrocytes such as CD40 and
CD86.182 Moreover, IL-­
21R−/− B cells are predominantly located
within the FDC network, further suggesting that IL-­21 signaling is
required to properly instruct the centocyte to centroblast transition.
By contrast, IL-­4-­dependent signaling appears to have a greater con-
tribution to the proper formation of the centrocyte profile. Although
−/−
STAT6 GC B cells have a broadly normal spatial distribution within
their smaller GCs, albeit with a smaller percentage of centrocytes,
the CXCR4lo centrocytes that form aberrantly underexpress CD86.
Together, these results suggest that newly generated centroblasts
formed without prior IL-­4 exposure might be compromised in their
ability to recapitulate the complete centrocyte program. This is con-
sistent with a study by Turqueti-­Neves and coworkers that examined
the response of IL-­4-­deficient mice to helminth infection and ob-
served a preponderance of DZ phenotype GC B cells that could be
corrected by the introduction of IL-­4-­sufficient cells in BM chime-
ras.194 Together, these intriguing studies raise more questions than
they answer. Can these signals be received independently during se-
quential cell contacts with Tfh cells that differ in their cytokine pro-
file? Are DZ centroblasts engaging Th cells within that compartment?
Although more work is needed to address these and other questions,
it is clear that the programming of GC B cells capable of responding
to both cytokines is different from that obtained with one but not
the other.
5 | E S TA B LI S H M E NT O F
I NTR A FO LLI CU L A R G C S A N D TH E
CO E VO LU TI O N O F FO LLI C U L A R S TRO M A L
C E LL N I C H E S
The cellular composition of tissue compartments effectively creates
microclimates that can be quite different. The cell types residing in
each compartment, together with the cytokines and chemokines
−/−
they secrete, likely shape the nature of lymphocyte activation, pro-
liferation, and differentiation.10,110 Differences in local accessory
cells ensure that B cell experiences within the molecular milieu of
one compartment will be different from those experienced in an-
other compartment. For example, the composition of the stromal cell
population, their activation status, and any molecular products could
potentially create a microenvironment that fosters modes of B cell
growth or effector lineages that are distinct from those occurring
elsewhere. Although stromal communities may delineate the bounda-
ries of neighborhoods defined by characteristic chemokine compo-
sitions, they are themselves capable of change under inflammatory
conditions.212 Here, we review the progression of events that occur
within the follicle as Tfh and GC B cells arrive, stromal cell popula-
tions evolve, and the unique compartments of the mature GC are
established.

F I G U R E   3   Stages of GC maturation and associated tissue niches. (A) Schematic representation of the stages of germinal center (GC)
maturation. (B-­E) Immunofluorescence staining of popliteal lymph node (B) and spleen (C-­E) from C57BL/6 mice that received an adoptive
transfer of OVA-­specific T cells and antigen-­specific B cells enriched by immunomagnetic purification (StemCell Technologies). T cells
specific for OVA were obtained from spleens of OT-­II mice while either B1-­8+ Jκ−/− or MD4 mice served as donors for anti-­NP-­or anti-­HEL-­
specific B cells, respectively. Recipients were immunized after transfer with 50 μg of NP-­OVA/CFA in the footpad (lymph node, panels
B), NP-­OVA/alum injected i.p. (spleen, panels C, E), or HEL-­OVA/alum (spleen, panel D). (B) Popliteal lymph node histology time-­course
demonstrating GC maturation after immunization. Stages of maturation represented include the unimmunized follicle (d0/naïve), maturation
of the FDC network and light zone expansion (day 4), centroblast increase, zonal segregation and follicle swelling (day 5), and a mature GC
(day 10). Sections were stained to highlight CD4+ T cells (blue), IgD+ non-­responding B cells within the follicular mantel (green), and CD35+
FDC network (red). (C) Histology showing a maturing GC (day 6) stained to highlight IgD+ naïve B cells (grayscale), PNA+ GC cells (green),
and the distribution of sonic hedgehog (SHH, red) in relation to the FDC network stained with CD35 (blue). (D) Representative histology of
splenic GCs demonstrating initial differentiative events shortly after the arrival of Ag-­specific B cells to the follicle interior (day 3). Sections
were stained to highlight GFP+ Ag-­specific B cells (green), CD35 FDCs (blue), and Bcl6 (red) to identify GC committed B cells, and IgMa to
assess the level of cytosolic immunoglobulin. Arrows point to B cells within the FDC network that are cIg hi (consistent with ASC effector
lineage), Bcl6hi (GC lineage), and Bcl6neg cIg lo (of an undefined alternative fate). (E) Histology showing GFP+ Ag-­specific B cell interaction
with dendritic cells (DC) at the T-­B border (day 6). Section was stained for CD4+ T cells (blue), B220+ B cells (red), GFP+ Ag-­specific B cells
(green), and CD11c+ DC cells (grayscale). Higher-­magnification images of the T-­B border demonstrate the ability of GC B cells, DCs, and T
cells at the T-­zone border to directly engage each other
HABERMAN et al. |
      17

(A) Stages of GC maturation

(B) Intrafollicular GC expansion

CD4 IgD CD35

d0 d4 d5 d10
(C) FDC network differences (D) ASC within nascent GCs
IgD PNA CD35 SHH CD35
GFP
Ig
Bcl6 CD35 Ig Bcl6

d3.5 Ig
(E) DZ-T zone interface

d6 CD4 GFP IgD CD11c

dendritic than is typically observed within mature GCs (Figure 3A

5.1 | Early intrafollicular events and FDC maturation
Follicular dendritic cells are derived from a stromal cell subset that
uniquely differentiates within B cell follicles and are the primary
97,213,214
source of CXCL13 in this zone of primary lymphoid tissues.
Within lymphoid tissue of naïve mice, FDCs can be identified as
uniquely expressing the long variant of the complement receptor
14
CR1 (CD35) within follicles. They are centrally and sparsely dis-
tributed within primary B cell follicles and are substantially less
and B). Immunization promotes the furthered development of FDCs
from perivascular-­associated stromal cells, which then protrude long
dendritic extensions forming an extensive network of dendrites.9
Immunization can also elicit the formation of FDC-­like cells from the
MRCs that descend from the SCS of LNs or marginal sinus lining cells
of spleens, and hence contribute to the CD35+ CXCL13-­producing
dendritic network closer to the outer follicle sinus.98,99 Both a nu-
merical increase and furthered differentiation occurs with these
 |
18       HABERMAN et al.
MRC-­derived FDCs, known to uniquely produce RANK-­L within the
follicle.99
After Tfh and pre-­GC B cells have further differentiated at the
interfollicular zone and T-­B border, they are observed to migrate
directly from that region toward the follicle interior, with the mi-
gration of disengaged cognate T cells occurring independently and
preceding that of GC B cells.126 Responsiveness to CXCL13, down-
regulation of EBI2 and CCR7 all promote this initial intrafollicular
movement.10,103,104,129 Once within the follicle, Bcl6hi B cells are pri-
marily found in the outer follicle, the upper follicle more distal to the
T-­cell zone, and appear there typically 3-­4 days postimmunization in
126,127
non-­infectious experimental systems.
FDCs undergo a form of activation after immunization that is
influenced by TLRs and NF-­kB signaling pathways, as well as im-
mune complex binding. 215-218 This activation results in an increase
in the expression of numerous proteins that can play a role in GC
B biology, including Fc receptor FcγRIIb, complement receptors
215,217-219
CD21/35, ICAM1, and VCAM1. FcγRIIb together with the
complement receptors CD21/CD35 mediate the bulk of the antigen
retention by the binding to proteins in immune complexes or tagged
with activated complement components. 220 After immunization,
FDCs also increase their production of Mfge8 that will promote the
phagocytosis of emerging apoptotic B cells by tingible body macro-
phages.7,221 FDC activation and dendrite extension is similar in tim-
ing to the arrival of Tfh and GC B cells to the follicle interior. Notably,
Tfh cells when first arriving to the follicle interior within LNs can
first be found primarily in contact with the centrally located and un-
derdeveloped CD35+ FDCs, but are also found in the follicular space
above nearer the SCS (Figure 3B).126,127 Although coincident, there
is only in vitro evidence to suggest that Tfh cells can influence FDC
activation222; there is no evidence to date in vivo. However, the TLR-­
dependent activation of FDCs suggests that some aspects of their
furthered differentiation can take place after exposure to adjuvant
216
alone.
As the GC matures, FDC network differences become more ap-
parent. ICAM, FCγRIIb expression,223 and sonic hedgehog (SHH) ex-
pression (Figure 3C)224 by FDCs are more evident within the GC LZ,
and in particular at the LZ-­DZ interface, than is observed with the
FDCs within the FM more proximal to the SCS. Given the positioning
of MRC-­derived FDCs closer to the SCS of LNs, it may be that their
99
contribution to the FDC network found within the FM is larger. The
functional differences between these FDC zones suggest that the na-
ture of the cellular interactions with lymphocytes within these distinct
domains could differ. For example, SHH found within GC LZs is known
to influence self-­renewal and cell-­fate determination in other tissue
compartments, including as hematopoietic niches, hair follicles, and
thymocyte selection.225-228 In support of the idea that SHH could in-
fluence LZ centrocytes, it has been reported that a subset of GC B cells
express both of the required components of the Hedgehog (Hh) recep-
224
tor, Ptc and Smo. Interestingly, expression of Smo can be increased
with CD40 or BCR signaling,224 suggesting that successful engagement
with stimulatory antigen and/or Tfh cells would empower hedgehog
signaling. Hedgehog signaling in GC B cells promotes their viability in
vitro and reduces Fas-­induced apoptosis,224 providing further evidence
that within the GC LZ both FDC-­derived and Tfh-­derived factors can
promote protection from the apoptosis-­prone state of GC B cells.215,229
5.2 | B ­cell differentiation within the newly
formed LZ
Studies that have examined the formation of BM-­resident long-­
lived plasma cells have observed that the plasma cells that per-
sist are predominantly generated after the peak of the GC
response.125,230 However, there is evidence of B cell differentia-
tion toward this effector lineage within the FDC network, even
at this very early stage. In addition to Bcl6+ GC B cells, antigen-­
specific B cells with large amounts of cytosolic immunoglobulin
(cIg) can be seen centrally placed in follicles and adjacent to FDCs
(Figure 3D).126 It remains unclear whether the interactions with
accessory cells within the still evolving LZ microenvironment are
sufficient to instruct the transcriptional program that is needed
for longevity in plasma cells. 231
At this same place and time, within the emerging GC LZ, re-
sponding B cells lacking both Bcl6 and cIg can also be observed,
suggestive of an alternative lineage fate such as a GC-­d erived
memory B cell. Although the lineage fate of such Bcl6neg antigen-­
specific B cells remains unclear, and whether they have the ca-
pacity to persist long term remains unknown, these observations
nevertheless suggest that not all GC B cell progeny are committed
to Bcl6 expression and self-­renewal in this arena even during this
stage of LZ expansion. Although Tfh cells are already in residence
at this point in time, it is intriguing that the first events are not
entirely devoted to centroblast formation. Given that the produc-
tion of long-­lived memory B cells is the predominant effector cell
output of younger GCs that persists,125,232 it is tempting to spec-
ulate that the unique conditions of this stage may contribute to
that lineage-­f ate preference. There are multiple differences within
newly formed GCs (days 3-­5 with most immunization regimes): (a)
FDCs have more recently undergone activation, (b) antigen depos-
its may be less likely initially to be in complexes with high-­affinity
antibody, possibly enabling more processes reliant on activated
complement, 220,233 (c) newly arrived Tfh cells are more prone to
produce IL-­21 than IL-­4 than at later stages in GC maturation, 23
and (d) LZ-­resident B cells have just transitioned from the perifol-
licular pre-­G C program and could therefore theoretically not have
yet obtained the centrocyte program or higher affinities that are
typical of a late time point. 55,234
5.3 | B ­cell expansion and effector differentiation at
peripheral sinuses
When colonization of the FDC network by GC B cells has just begun,
B cell differentiation toward other non-­GC lineages is also apparent
by histology at other peripheral arenas. Strikingly, at this very early
stage of the immune response, expanded populations of antigen-­
specific B cells form immediately under the SCS of LNs126,127 and
HABERMAN et al.
at splenic marginal sinuses. 235,236 This phenomena is short lived and
quickly diminishes as the GC response progresses. During this early
phase, responding B cells at the SCS were seen to exit follicles via the
subcapsular lumen of LNs during intravital imaging, but importantly,
the reverse was not true—antigen-­specific B cells near the SCS were
not observed to enter the follicle via that route, strongly suggest-
ing that proliferative B cells at the SCS do not contribute to the
GC reaction.126 In this study, Bcl6 protein was not apparent within
B cells at that location as assessed histologically. This is in contrast
to the results of another histologic study that discerned a temporal
relationship of first a predominance of Ki67+ GL7+ B cells adjacent to
the marginal sinus, followed by an appearance of aggregated GL7+
B cells in follicle interiors. Here, GL7 expression was presumed to be
a marker uniquely expressed by GC destined B cells. Although GL7
expression appears restricted to B cells within GCs at mature time-
points,87 it is important to note that it is also known to be expressed
by LPS-­stimulated B cells in vitro, including by B cells forming ASC89
+ 237
as well as by IgM early memory B cells. In the Coffey et al
study, Bcl6 levels were assessed by qRT PCR of mRNA. However,
Bcl6 can be transcribed with little translation to protein in some cell
types.162 Thus, the difference in these studies vis a vis GC B cell for-
mation might therefore relate to the well-­known post-­transcriptional
regulation of Bcl6. 238-240
There is some evidence of ASC development among activated
B cells at LN SCS. Some SCS-­adjacent B cells have copious amounts
of cIg light chain, consistent with commitment to a ASC lineage.
However, in another study, B cells adjacent to splenic marginal sinus
lacked the CD138 expression that is characteristic of late-­
stage
plasma(blast) cell formation and had not undergone class switch re-
combination to IgG. 235 Together the results of these studies remain
consistent with the idea that a subset of sinus lining B cells are in the
process of differentiating to IgM ASCs. In addition to the SCS, ASC
cell formation can also be seen at the T-­B border, and among the re-
126,241
maining cognate B and T cells within the interfollicular region.
Consistent with this, prdm1-­expressing ASC B cells have also been
observed to move from deeper perifollicular locations toward med-
ullary sinuses.90 The lineage fates of the non-­A SC B cells at these
alternative locations remain unclear, but given the absence of Bcl6
+
protein and prior reports of very early IgM memory B cells that are
237
GC independent, it is likely that a subset is destined to become
early memory B cells or alternative B cell effectors.123,125
5.4 | Zonal segregation and DZ evolution
Depending on the immunogen and mode of immunization, the emer-
gence of a discernable DZ may not occur for another day or two. This
zonal segregation requires, by definition, the presence of GC B cells
outside of the FDC network, but as described below there are sev-
eral other unique features of the DZ compartment that evolve dur-
ing the course of the response. One key element of the centroblast
program is the increased expression of CXCR4, which enables centro-
blast migration to CXL12-­producing stroma initially found most proxi-
mal to the basement of the follicle, near the T-­B border.10,33,242 These
|
      19
CXCL12-­producing reticular cells (CRC) also have a reticular morphol-
ogy, although the dendritic extensions lack the high levels of CD35 ex-
pression and complement binding that are distinctive for FDCs.31,32,242
Interestingly, in some but not all GCs, there are DZ areas that are not
filled by a CRC network.242 The DZ stromal cells without CRC features
are as yet uncharacterized, but are found closer to the DZ/LZ inter-
face and at this position could potentially shelter GC B cells from the
influences of both CRC and FDCs. In this regard, the distribution of
freshly minted centroblasts is intriguing. The emerging centroblasts
typically form both a collection immediately under the FDC network
as well as a loose column that stretches toward the T-­zone border
(Figure 3B).141,182 This is consistent with chemotaxis toward CXCL12
as the primary drive for movement out of the FDC zone.10
CRC can differ phenotypically and morphologically within the
DZ, perhaps relating to differences in either their derivation or mode
of activation. Although fibroblastic reticular cells (FRCs) similar to
T-­zone FRC are only peripherally present at the edge of the B cell
235
follicle in naïve LNs, after immunization the expanding GC swells
the follicle, causing both IgD+ and GC B cells to shift past the large
cortical lymphatic sinuses present at the naïve border and into the
T-­zone FRC-­rich area.100,105,106 Results presented by Mionnet et al100
are consistent with the idea that at least some DZ stroma may de-
rive from T-­zone FRC cells. In this way, new microenvironments are
created by evolving stromal cells thereby forming a novel venue for
events that might not have otherwise been possible, in this case an
126
avenue bringing DZ GC B cells in close contact with the T-­zone bor-
der. Like other T-­zone FRC, the stromal network newly infiltrated by
the expanding follicle also exhibits a collagen dense core typical of
FRC-­ensheathed lymph conduits, 243,244 a feature that is less apparent
among the more centrally positioned CRC within mature DZs.32,242
Given that CRCs are also found within primary non-­reactive B cell
follicles, 242 it seems possible that CXCL12-­expressing stroma within
DZ compartments can derive from more than one cell type.
In addition to the blasting centroblasts, GC DZs also harbor fol-
licular T cells as well as DCs (Figure 3B). Not all follicular T cells are
located in the LZ. They can also be found within the center of DZs as
well as at the outer edges of DZs adjacent to the FM, albeit in lower
densities (Figure 3B). 21,245 Although little is known about how these
CD4+ T cells and DC subsets might differ from their counterparts
at the T-­B border and GC LZ, there is evidence to suggest that Tfh
cells producing IFNγ or IL-­21 can localize to the DZ outer edge within
the follicle. 21,23 Follicular regulatory T cells (Tfr) have also been ob-
served within DZs. 245 Such DZ-­resident T cells are in a unique posi-
tion to influence centroblast behavior, either favorably as they might
engage Tfh cells within the LZ, or in the case of DZ Tfr cells influenc-
ing centroblast self-­renewal or differentiation to long-­lived memory
or plasma cells. 246-248
5.5 | Follicular Mantle
As the GC expands, GC B cells form a large egg-­shaped aggregate that
displaces non-­responding B cells, causing them to form the follicu-
lar mantle. Intravital imaging has demonstrated that non-­responding
 |
20       HABERMAN et al.
B cells are not excluded by a physical barrier and can move freely
70,72
through GCs. Although early stages of GC development show
extensive admixing with IgD+ non-­cognate B cells (Figure 3B), this is
uncommon within mature GCs. The follicular mantle harbors more
than just non-­responding B cells. In addition to the sinus-­proximal
FDCs described above, a distinct subset of Tfh cells can also be found
within FMs. The follicular mantle surrounding mature GCs harbors Tfh
cells that are phenotypically and functionally distinct, with a lower
22,122
expression of IL-­4. After the resolution of a primary response,
both memory B cells and memory Tfh cells can be found within FMs, a
positioning that is influenced by their EBI2 expression.122,249,250
Theoretically, this compartment could influence the differen-
tiative state of antigen-­specific B cells that are leaving the GC via
this route. As cells within this arena are less likely to be in active
cell cycle, this environment may be more conducive to gaining a
quiescent state, sheltered from the cellular interactions that pro-
mote a centroblast program and their self-­renewal. It is import-
ant to note that this is not the predominant route of exit for the
prdm1-­expresing ASCs leaving GCs, which largely leave via the
GC outer zone through the DZ base due to their downregulation
82,90,251-253
of CXCR5 and increased responsiveness to CXCL12.
However, the FM could be the preferred exit route of GC-­d erived
memory B cells, as has been shown for Tfh memory cells during
122
secondary responses. CCR6 expression has been identified as
a distinctive feature of memory B cells, 254,255 including those ob-
256
served to be completing their differentiation within GC LZs.
ligand for CCR6, CCL20, is known to be produced by some ep-
ithelial cell types and importantly by lymphatic endothelial cells
lining the subcapsular sinus of LNs near B cell follicles, the latter
shown to be key to the localization of IL7Ra+ CCR6hi innate-­like
lymphocytes. 257,258 FM non-­responding B cells are also CCR6hi, in
sharp contrast to CCR6lo GC B cells. However, naive B cells lack
responsiveness to CCL20 despite expressing the receptor for this
chemokine, unlike CCR6hi memory B cells which chemotax well
255
to CCL20. Although memory B cells have been reported to be
positioned within lymphoid tissues at marginal zones, perifollicular
zones, and some follicles after the resolution of response, 255,259,260
two studies have reported a predominant FM location for Ki67-­
memory B cells in spleens and LNs. 249,250 Although the above
studies support the idea that memory B cells primarily exit via fol-
licle adjacent sinuses, it is worth noting that CCR6hi memory B
255
cells are also responsive to CXCL12.
5.6 | The unique niche of the DZ-­T zone border in
mature GCs
The follicular mantle is thin to non-­existent at the true base of the DZ
in mature GCs. In cross sections of mature GCs that are central and
en face, IgD+ B cells can be infrequent or absent at the base of the
DZ. This DZ-­T zone interface is commonly observed in splenic and LN
GCs when canvassed in their entirety via serial sections (Figure 3E,
Haberman lab unpublished data).32,82,242,261 The DZ-­T zone interface
can be quite extensive in larger GCs and could theoretically allow DZ
GC B cells to interact with cell types that are not present in other GC
compartments.
This arena of mature GCs is poorly understood but clearly has
the potential to be an influential venue for self-­renewing cen-
troblasts as well as GC-­d erived long-­term plasma cells. Maturing
plasmablasts can be found at this location, 82,251,262 and in a re-
cent study found to be in association with CD157hi podoplanin+
FRCs as early as 6 days postimmunization. 241 Similar to the CRC
cells within the DZ, the reported CD157hi FRC at the DZ base of
young GCs also express CXCL12, but appear to lack dendritic ex-
tensions into the DZ. 241,242 An intriguing analysis of LN stromal
cells by single-­cell RNAseq further suggests that FRC at the T-­zone
interface with B cell follicles is heterogenous, and may include
both CCL19lo and CCL19hi subsets, as well as activated subsets
responding to inflammation.109
There are several features to this unique niche that have the po-
tential to influence centroblasts and other DZ-­resident cell types at
this DZ-­T-­zone border. Here, there are opportunities for direct con-
tact with perifollicular Th cell and DC subsets (Figure 3E), as well as
Tregs.245 There is also the potential for fresh antigen delivery via in-
coming local lymph within adjacent sinuses.106,243,263 Scavenging APCs
could theoretically participate in antigen acquisition from local sinuses,
similar to DCs probing lymphatic sinuses or FRC junctures,244,264 and
provide antigen processing and presentation to perifollicular Tfh cell
present at the DZ-­T border. In this regard it is interesting to note that
A some DCs have a specialized capacity to retain and present antigens in
native form without degradation, theoretically permitting BCR cross-
linking of centroblasts in direct contact with DCs presenting incom-
ing lymph-­borne antigen.115,265 Thus, at this niche, DZ GC B cells may
have access to antigen to process and present, as well as APCs and
T-­cell help, which in toto could be sufficient for any of the proposed
mechanisms for GC B cell selection.33,43,45,59 Consistent with this
niche playing a role in DZ B cell dynamics, movement to and from the
DZ-­T-­zone border has been observed by intravital imaging.70 Future
work in this arena is very much needed to gain insight into this poorly
understood niche.
6 | CO N C LU S I O N
GC development is a complex series of events that requires that
cognate B and T cells periodically engage during discreet stages in
their differentiation and at distinct locations within tissue. While GC
B- cell development begins at the periphery of B cell follicles, this
line of differentiation is progressive and is characterized by step-­
wise changes in transcription and phenotype over the course of
multiple cell divisions. GC B cell development can be paused at an
intermediate stage of differentiation when conditions are not quite
right, permitting the refinement of the immune response when an-
tigen is in excess, or when the differentiation of regional activated
T cells has not sufficiently progressed. Under conditions when re-
cently activated CD40L+ CD4+ T cells are present, but IL-­21 and IL-­4
are lacking, pre-­GC B cells will transiently persist at perifollicular
HABERMAN et al.
locations. This is in part regulated by a stage-­specific effect of CD40
signaling; while an early phase in GC B cell development is depend-
ent on CD40 signaling, the subsequent phase is refractory to CD40
agonism and likely involves a transient abstention from T-­cell help
at the periphery of B cell follicles. When IL-­21 or IL-­4 signaling also
occurs, then GC B cell maturation is allowed to progress to the next
stage that empowers follicular entry, but only if CD40 agonism is
transiently avoided. The migration of Tfh cells into the follicle inte-
rior before GC B cell arrival might therefore help at multiple stages
and locations by: (a) vacating the perifollicular sites and thereby re-
ducing the chance of CD40L stimulation, and (b) entering into the
follicle in advance, immediately ensuring that centrocyte and centro-
blast instruction occurs properly and sustainably, events that are re-
liant on both IL-­21 and IL-­4. Thus, early B and T cognate interactions
at the follicle periphery change in quality as they co-­evolve, critically
influencing when and how intrafollicular GCs are established.
All these early events are temporally and spatially choreographed
by the serial movements of cognate B and T cells to different and dis-
creet tissue locales. The stroma at these tissue destinations are them-
selves influenced by the evolving immune response in their arena.
Together these interactions help to ensure that activated B cells re-
ceive the correct signals for their stage of GC B cell differentiation
and coordinate the timing and extent of that differentiation. Finally,
we conclude that it is advantageous for GC B cell differentiation to
be staged and slowly progressive in that this creates checkpoints that
allow for the initial creation of GC precursors, without committing to
an energy-­intensive, and potentially misdirected and unproductive GC.
AC K N OW L E D G E M E N T S
We thank Jaymin Patel for skillful technical assistance with IF his-
tology. This work was supported by National Institutes of Health
NIAID grants R01AI080850 and R21AI101704 and NIAMS
Rheumatic Diseases Research Core Centers grant P30AR053495.
T. Z. was supported by a fellowship by the Canadian Institutes of
Health Research. S. M. K. is funded by an Operating Grant from the
Canadian Institutes of Health Research and was the recipient of
the Garrett Herman endMS Research and Training Network Career
Development Award from the Multiple Sclerosis Society of Canada.
C O N FL I C T O F I N T E R E S T
The authors certify that there is no actual or potential conflict of
interest in relation to this article.
ORCID
Ann M. Haberman  http://orcid.org/0000-0001-5168-8229
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How to cite this article: Haberman AM, Gonzalez DG, Wong P,
Zhang T, Kerfoot SM. Germinal center B ­cell initiation, GC
maturation, and the coevolution of its stromal cell niches.
Immunol Rev. 2019;288:10–27. https://doi.org/10.1111/
imr.12731