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Immunity
Review
T Follicular Helper Cell Differentiation,
Function, and Roles in Disease
Shane Crotty
1,
*
1
Division of Vaccine Discovery, La Jolla Institute for Allergy and Immunology, La Jolla, CA 92037, USA
*Correspondence: shane@lji.org
http://dx.doi.org/10.1016/j.immuni.2014.10.004
Follicular helper T (Tfh) cells are specialized providers of T cell help to B cells, and are essential for germinal
center formation, affinity maturation, and the development of most high-affinity antibodies and memory B
cells. Tfh cell differentiation is a multistage, multifactorial process involving B cell lymphoma 6 (Bcl6) and
other transcription factors. This article reviews understanding of Tfh cell biology, including their differentia-
tion, migration, transcriptional regulation, and B cell help functions. Tfh cells are critical components of many
protective immune responses against pathogens. As such, there is strong interest in harnessing Tfh cells to
improve vaccination strategies. Tfh cells also have roles in a range of other diseases, particularly autoimmune
diseases. Overall, there have been dramatic advances in this young field, but there is much to be learned
about Tfh cell biology in the interest of applying that knowledge to biomedical needs.
Introduction
There has been a great deal of recent activity in the study of T
follicular helper (Tfh) cells. While the first evidence of Tfh cells
was reported in human lymphoid tissue more than a decade
ago, much of the interest in Tfh cells traces its origins to the
identification of Bcl6 as an essential transcription factor in
CD4
+
T cells for Tfh cell differentiation and the development of
germinal centers (GCs) (Johnston et al., 2009; Nurieva et al.,
2009; Yu et al., 2009). The field of Tfh cell biology has now
exploded with activity, examining everything from the biochem-
istry of transcription factors involved in programming Tfh cell dif-
ferentiation to the cellular biology of Tfh cell-mediated selection
of germinal center B cells, and examining important roles of Tfh
cells in biological processes as diverse as vaccine-elicited im-
mune responses, chronic autoimmune diseases, and even roles
of Tfh cells in protective immunity in human cancers. This article
reviews our understanding of Tfh cell differentiation, molecular
biology, and function and discusses the most recent advances
in these areas, as well as the complexities of Tfh cell biology.
In addition, a new conceptual model is introduced to explain
the relationship between Tfh cell and other CD4
+
T cell differen-
tiation programs. For an oral presentation of the review, see
Movie S1 available online.
Stages of Tfh Cell Differentiation
Tfh cell differentiation is a multistage, multifactorial process.
There is no single event that defines Tfh cell differentiation, unlike
T helper 1 (Th1) cell differentiation, for instance, which can be
fully induced by interleukin-12 (IL-12) exposure in vitro or in vivo.
Instead, Tfh cell differentiation is a multistep, multisignal process
that also accommodates a significant amount of heterogeneity.
The canonical Tfh cell differentiation process starts at initial den-
dritic cell (DC) priming of a naive CD4
+
T cell (Goenka et al., 2011)
(Figure 1A). The CD4
+
T cell undergoes a cell-fate decision within
the first few rounds of cell division (Choi et al., 2011; 2013b). If the
chemokine receptor CXCR5 is expressed, the early Tfh cell will
migrate to the border of the B cell follicle and undergo further
Tfh cell differentiation. If the cell instead receives Th1, Th2, or
Th17 signals (Figure 1), then the CD4
+
T cell follows a Th1,
Th2, or Th17 cell differentiation program, including upregulation
of chemokine receptors for inflammatory chemokines that will
drive the effector cell to exit the lymphoid tissue and traffic to
the site of infection or inflammation.
Early Tfh cell differentiation (the DC priming phase) is regulated
by IL-6, inducible costimulator (ICOS), IL-2, and T cell receptor
(TCR) signal strength in mouse models. TCR signal strength
can bias T cell differentiation in vivo (Tubo et al., 2013), but a sin-
gle naive mature T cell can give rise to multiple different differen-
tiated effector cell types upon stimulation and proliferation,
demonstrating that non-TCR and TCR signals combine to deter-
mine T cell differentiation fates. CD4
+
T cells possessing TCRs
with high affinity preferentially differentiated into Tfh cells in a
pigeon cytochrome C (PCC) model (Fazilleau et al., 2009), but
not a Friend virus infection (Ploquin et al., 2011). Utilizing a range
of systems, it was found that TCR: major histocompatibility com-
plex-II (MHCII) dwell time is a more accurate predictor of cell-fate
preference, with a nonlinear relationship (Tubo et al., 2013), such
that there was no simple relationship between TCR signal
strength and Tfh cell differentiation. IL-6 is the earliest non-
TCR signal involved in initiation of Tfh cell differentiation. IL-6
signaling through IL-6 receptor (IL-6R/gp130) transiently induces
Bcl6 expression by newly activated CD4
+
T cells (Nurieva et al.,
2009). Bcl6 is necessary for early CXCR5 expression in multiple
models (Choi et al., 2011; 2013a; Pepper et al., 2011). In the
absence of IL-6 an early defect in Tfh cell differentiation is
observed (Choi et al., 2013a). The DC type responsible for initi-
ating Tfh cell differentiation is unknown. Most likely, there are
multiple Tfh cell differentiation pathways and there is no single
DC type responsible. Instead, multiple DC and monocyte types
can prime Tfh cell differentiation in different conditions (Balles-
teros-Tato and Randall, 2014). Many DC types are robust
producers of IL-6. Prdm1
/
DCs are hyperactive producers of
IL-6, resulting in spontaneous Tfh cell and GC development
in vivo (Kim et al., 2011). IL-6 can also be a signal for Th17
cell differentiation, and therefore it is assumed that IL-6, in
Immunity 41, October 16, 2014 ª2014 Elsevier Inc. 529
combination with different signals, is involved in Tfh cell versus
Th17 cell differentiation. Interestingly, no increase in Th17 cells
was seen in hyper-IL-6-producing mice, in contrast to the in-
crease in Tfh cells. IL-1 is an important driver of Th17 differenti-
ation, whereas ICOS is important for Tfh differentiation (Choi
et al., 2011; Nurieva et al., 2008). ICOS has roles in both Tfh
cell differentiation and migration, and there are data supporting
a synergistic role of ICOS and IL-6. The importance of ICOS is
highlighted by the multiple ways in which ICOS signaling is regu-
lated. Roquin inhibits ICOS, and combined loss of Roquin1 and
Roquin2 results in spontaneous Tfh cell and GC development
(Pratama et al., 2013; Vogel et al., 2013). In addition, the miR-
1972 complex is necessary for Tfh cell differentiation, and it
works, in part, via dampening the PI(3)K inactivating phospha-
tases PHLPP2 and PTEN, which are inhibitors of ICOS signaling
(Baumjohann et al., 2013; Kang et al., 2013). IL-2 signaling is
another major regulator of Tfh cell differentiation. IL-2 is a potent
inhibitor of Tfh cell differentiation (Ballesteros-Tato et al., 2012;
Johnston et al., 2012) and can act very early during T cell priming
(Johnston et al., 2012). Thus, the interplay between IL-6, ICOS,
IL-2, and TCR signaling orchestrates early induction of mouse
Tfh cell differentiation during DC priming via control of CXCR5,
Bcl6, and other targets.
The second stage of Tfh cell differentiation occurs when the
T cell interacts with antigen-specific B cells in the follicle, interfol-
Figure 1. Overview of Tfh Cell
Differentiation
(A) Stages of Tfh cell differentiation, highlighting
roles of migration-associated molecules.
(B) Signals in CD4 T cell differentiation. A simplified
model of CD4 T cell differentiation pathways,
showing transcription factors and inducing fac-
tors, highlighting apparent differences between
human and mouse Tfh cell differentiation.
licular zone, or the T-B border. Much of
Tfh cell differentiation and function is
tightly interconnected with the microana-
tomical geography of the T and B zones
of the lymph node (LN) and spleen. The
early Tfh cells colocalize with B cells
because they express CXCR5, downre-
gulate C-C chemokine receptor type 7
(CCR7) (the primary chemotactic recep-
tor for the T zone), and downregulate P-
selectin glycoprotein ligand 1 (PSGL1),
which is thought to anchor T cells to
CCL19 and CCL21 decorating the T
zone extracellular matrix (Figure 1A). Tfh
cells have a highly symbiotic relationship
with B cells, and B cells are required for
Tfh cell development under almost all
conditions (Crotty, 2011). ICOS is a costi-
mulatory molecule, but it has been
recently demonstrated that ICOS-ICOS
ligand (ICOSL) binding also induces
directional migration of CD4
+
T cells,
which can play an important role in proper
localization of the early Tfh cells to the B
cell follicle (Xu et al., 2013). B cells serve both as antigen-pre-
senting cells (APCs) and as a source of ICOSL (Choi et al.,
2011; Haynes et al., 2007; Nurieva et al., 2008). B cells rapidly
become the primary APCs available in a LN during an acute
infection or immunization because mature DCs last for only a
few days before dying, whereas the antigen-specific B cells un-
dergo geometric replication. Antigen presentation is critical,
because unlike effector CD8 T cells, antigen-specific CD4
T cells require antigen recognition for virtually every cell division
(Choi et al., 2013b; Obst et al., 2005; Yarke et al., 2008).
The third stage of Tfh cell differentiation involves the GC
(Figure 1A). The GC is a distinct structure consisting of GC Tfh
cells, GC B cells, follicular dendritic cells (FDCs), macrophages,
and stroma. The majority of GC Tfh cells can be observed
to possess a canonical Tfh cell differentiation program. The
majority of GC Tfh cells are CXCR5
hi
PD1
hi
Bcl6
hi
Maf
hi
SAP
hi
.
They are also PSGL1
lo
CD200
+
BTLA
hi
CCR7
lo
. The canonical
secreted Tfh cell molecules are C-X-C motif chemokine 13
(CXCL13), IL-21, and IL-4 (Crotty, 2011; Kroenke et al., 2012;
Liang et al., 2012; Linterman et al., 2011). These GC Tfh cell sur-
face proteins, transcription factors, and secreted molecules are
well conserved across in vivo conditions and species. GC Tfh
cells can be readily identified in mice, humans, and nonhuman
primates as CXCR5
hi
PD1
hi
Bcl6
hi
CD4 T cells. The biology of
GC Tfh cells is strongly associated with changes in several
530 Immunity 41, October 16, 2014 ª2014 Elsevier Inc.
Immunity
Review
chemokine receptors and related molecules. GC Tfh cells have
very high expression of CXCR5, low expression of CCR7,
elevated C-X-C chemokine receptor type 4 (CXCR4), low sphin-
gosine 1-phosphate 1 receptor (S1P1R), and very low amounts
of PSGL1. Loss of Epstein-Barr virus-induced G protein coupled
receptor 2 (EBI2) expression is notable because the chemoat-
tractant ligand for EBI2 is present in the B cell follicle, but not
the GC environment. Reduction of EBI2 expression by both
GC B cells and GC Tfh cells is important for their proper localiza-
tion to GCs (Hannedouche et al., 2011). In addition, adhesion
molecules play an important role on GC Tfh cells, regulating their
interaction with GC B cells and their localization. The signaling
lymphocyte activation molecule (SLAM) family receptors
SLAMF6 (also known as Ly108 and NTB-A), CD84, and SLAM
are all self-ligands differentially expressed on GC Tfh cells and/
or GC B cells. SLAM-associated protein (SAP), the product of
the Sh2d1a gene, is an SH2-domain adaptor protein that binds
to the cytoplasmic tails of SLAM family receptors and is specif-
ically upregulated in GC Tfh cells. SAP expression is essential for
GC Tfh cell development, GC development, and the generation
of the majority of memory B cells and memory plasma cells
(Hu et al., 2013). In the absence of SAP, Tfh cells have defective
adhesion to GC B cells and fail to be retained in GCs (Qi et al.,
2008); as a result, insufficient help is provided by SAP-deficient
Tfh cells to B cells. The functions of SAP are central to Tfh cell
biology, because loss of SAP is one of the few genetic mutations
in CD4
+
T cells that results in a complete loss of GC Tfh cells and
GC B cells in virtually all experimental settings. Surprisingly,
much of the importance of SAP is due to a requirement for
SAP to prevent powerful inhibitory signaling through SLAMF6
(Kageyama et al., 2012). SAP competes with the phosphatase
SHP-1 for binding to SLAMF6. With SAP bound, SLAMF6 trans-
mits positive signals within the Tfh cells, supporting adhesion
and help functions. With SHP-1 bound, SLAMF6 transmits
potent negative signals that truncate Tfh:B cell adhesion. Impor-
tant roles for SLAMF6 as a rheostat of cell:cell adhesion for lym-
phocytes have been shown for Tfh, natural killer T (NKT), CD8 T,
and NK cells. This also suggests that other adhesion molecules
are likely to be regulators of both GC Tfh cell differentiation and
function.
Once Tfh cells have differentiated into GC Tfh cells and pro-
vided help to GC B cells, they are not confined to the GC. GC
Tfh cells can exit GCs (Figure 1A). GC B cells are strictly confined
to a single GC, and the majority of GC B cells within a GC repre-
sent oligoclonal antigen-specific B cell clones undergoing hyper-
mutation and selection by Tfh cells. In contrast, the GC Tfh cells
can readily exit a GC and (1) transit to a neighboring follicle and
enter a different GC (Shulman et al., 2013), (2) temporarily reside
in the adjacent B cell follicle before re-entering the same GC
(Figure 1A), or (3) exit a GC and downregulate Bcl6 and develop
into a memory Tfh cell (Kitano et al., 2011; Shulman et al., 2013)
(Figure 2). Memory is discussed further in a section below.
Therefore, a CXCR5
+
Tfh cell outside of a GC might have already
been a GC Tfh cell and is in the process of transiting to a new GC,
or it might be a newly activated Tfh cell on its way to becoming a
GC Tfh cell, or it may have another fate, including memory forma-
tion or being a Tfh cell destined to provide help primarily outside
of a GC.
A canonical Tfh cell differentiation pathway was described
above, involving multiple signals, a multistage process, and
two different APCs. Alternative Tfh cell differentiation processes
exist. This is expected. There is not a single immutable Tfh cell
phenotype. It is quite clear that CD4
+
T cells have enormous
intrinsic heterogeneity. This is an important aspect of CD4
+
T cell biology, allowing the cells to adapt to a variety of environ-
mental conditions, locations, and needs. Some of this variability
is almost certainly stochastic and valuable for preventing path-
ogen evasion by virtue of its randomness and diversity. Th1,
Th2, Th17, and Th9 responses are selectively valuable for re-
sponses to specific categories of pathogens (e.g., Th1 cells in
response to viral infections) and are induced by pathogen-asso-
ciated molecular patterns (PAMPs) associated with a pathogen
category (e.g., viral RNA triggering of TLR7 and TLR8 causes
IL-12 production by DCs to instruct Th1 cell differentiation).
That conceptual framework does not hold true for Tfh cells. Anti-
body responses are valuable against almost all pathogens,
irrespective of whether they are viral, bacterial, fungal, or multi-
cellular parasites. Consequently, it is critical that the immune
system trigger Tfh cell inductive signals whenever any form of
pathogen is detected.
As a result of the need to trigger Tfh cell inductive signals in the
context of a wide range of potential pathogen assaults, multiple
redundant signals are likely involved. IL-6 is an example of this.
IL-6 is produced by DCs, macrophages, B cells, and a variety
of other cell types in response to a range of external and internal
PAMPs and damage-associated molecular patterns (DAMPs). In
the absence of IL-6, an early defect in murine Tfh cell differentia-
tion is observed, most likely due to a failure of IL-6 production by
the DCs priming naive T cells. Nevertheless, that defect is rapidly
Figure 2. Tfh Cell Memory Development
Memory Tfh cells develop over time and appear to develop from either Tfh cells
or GC Tfh cells. Memory Tfh cells exhibit phenotypic heterogeneity. Memory
Tfh cells that retain stable expression of low amounts of PD-1 (PD-1
lo
or PD-1
+
)
are more polarized and highly functional memory Tfh cells, when compared to
PD-1
neg
memory Tfh cells. Upon reactivation, memory Tfh cells predominantly
become Tfh cells and can go on to become GC Tfh cells, although some
memory Tfh cells can go on to become non-Tfh cells in a recall response.
Immunity 41, October 16, 2014 ª2014 Elsevier Inc. 531
Immunity
Review
compensated for by IL-21 or IL-27 in most cases (Batten et al.,
2010; Choi et al., 2013a; Eto et al., 2011; Harker et al., 2013; Kar-
nowski et al., 2012), though late roles of IL-6 for Tfh cells can also
be dramatic (Harker et al., 2011). A second example of multiple
alternative Tfh cell differentiation pathways involves ICOS (Wein-
stein et al., 2014; Xu et al., 2013). ICOSL was found to not be
essential on antigen-specific B cells (Xu et al., 2013), but a
follow-up study found that ICOSL is normally required on anti-
gen-specific B cells, but that can be overcome when large
numbers of antigen-specific B cells are transferred or in the pres-
ence of large amounts of antigen (Weinstein et al., 2014).
Given the importance of Tfh cells, one concept is that Tfh cell
differentiation is a default pathway for newly activated CD4
T cells. That concept appears to be incorrect. In vitro activated
CD4
+
T cells in unbiased conditions fail to acquire any of the
key features of Tfh cells such as CXCR5, Bcl6, SAP, or IL-21
expression (Eto et al., 2011).
Although much is now understood about the multiple stages of
Tfh cell differentiation and signals involved in the process, critical
knowledge is still lacking. Most importantly, understanding of Tfh
cell differentiation is still insufficient to establish a defined and
reproducible in vitro Tfh cell differentiation condition. This is
the single most serious knowledge gap in the field of Tfh cell
biology. Features of some partial aspects of Tfh cell differentia-
tion have emerged from in vitro studies. It is well established
that IL-6 is a potent inducer of IL-21 expression by activated
murine CD4
+
T cells. IL-6 also induces transient Bcl6 expression
(Eto et al., 2011; Nurieva et al., 2009). However, although IL-6
was initially reported to drive CXCR5 expression (Nurieva et al.,
2009), IL-6 does not induce significant amounts of CXCR5
mRNA or protein in most conditions (Eto et al., 2011; Liu et al.,
2014). Furthermore, IL-6 is not a good inducer of IL-21, Bcl6,
or CXCR5 expression by activated human CD4 T cells (Ma
et al., 2009; Schmitt et al., 2009). In contrast, IL-12 is a potent
inducer of IL-21 expression by human CD4 T cells, but not
murine CD4 T cells (Ma et al., 2009; Schmitt et al., 2009). Inter-
estingly, IL-6 deficiency results in a severe reduction in
CXCR5
+
Bcl6
+
early Tfh cells in vivo, with effectively no CXCR5
or Bcl6 expressing cells present 72 hr after an acute viral infec-
tion (Choi et al., 2013a). Nevertheless, constitutive Bcl6 expres-
sion in activated murine CD4
+
T cells is not sufficient to induce
CXCR5 expression in vitro (Liu et al., 2014). In contrast, constitu-
tive expression of Bcl6 in previously activated (CD45RO
+
) human
CD4
+
T cells does induce elevated CXCR5 in vitro (Kroenke et al.,
2012). A recent paper reports the most successful conditions yet
for human Tfh cell differentiation in vitro, with successful short-
term induction of CXCR5, Bcl6, and IL-21 in the presence of
transforming growth factor b(TGF-b) and IL-12 or IL-23 (Schmitt
et al., 2014). A contribution by TGF-bis surprising. Importantly,
these same conditions were not effective at inducing murine
CD4
+
T cells to differentiate into Tfh cells in vitro, implying a dif-
ference between the species. In vitro TGF-b+ IL-12 or TGF-b+
IL-23 generated human Tfh cells possessed enhanced B cell
help activity, indicating that TGF-bis important for human Tfh
cell differentiation and function.
Clues regarding human Tfh cell differentiation are also being
provided by analysis of humans with genetic deficiencies. The
importance of SAP and ICOS in human Tfh cell differentiation
and function was recognized a number of years ago (Crotty,
2011). Now it is also clear that IL-21R and STAT3, as well as
IL-12R and STAT4 are two pairs of proteins for which genetic
mutations are associated with loss of Tfh cells in humans (Ma
et al., 2012a; Schmitt et al., 2013). Importantly, it remains unclear
whether murine and human Tfh cell differentiation are regulated
by the same cytokines (Figure 1B), and therefore not all lessons
from one species may be applicable to the other, even though
the cell gene-expression program and Tfh cell functions are
highly conserved between the species.
Tfh Cell Memory
While Tfh cell memory was controversial (Ma et al., 2012b;
Marshall et al., 2011; Pepper et al., 2011), there are now a series
of clear studies demonstrating Tfh cell memory in both mice
(Choi et al., 2013b; Hale et al., 2013; Liu et al., 2012; Weber
et al., 2012) and humans (Bentebibel et al., 2013; Locci et al.,
2013), building upon earlier observations in both species (Cheva-
lier et al., 2011; Lu
¨thje et al., 2012; Morita et al., 2011). Memory
Tfh cells can be long-lived (Hale et al., 2013; Locci et al., 2013)
and are transferrable (Hale et al., 2013). Some of the hesitancy
regarding Tfh cell memory was based on an incorrect assump-
tion that GC Tfh cells were terminally differentiated and could
not leave GCs. In fact, GC Tfh cells regularly exit from GCs, as
discussed above. Upon leaving a GC, the Tfh cell acquires a
less activated, less polarized Tfh phenotype and can upregulate
IL-7Raand develop into resting memory Tfh cells (Choi et al.,
2013b; Hale et al., 2013; Kitano et al., 2011; Liu et al., 2012; Shul-
man et al., 2013; Yusuf et al., 2010)(Figure 1B). In addition, it is
not required that a Tfh progress through a GC Tfh state to
become a memory Tfh cell (He et al., 2013)(Figure 2). Memory
Tfh cells have a central memory phenotype and predominantly
reside in spleen, LNs, and bone marrow, and have the capacity
to recirculate in blood (Chevalier et al., 2011; Hale et al., 2013).
Approximately 20% of all human central memory CD4
+
T cells
are CXCR5
+
, demonstrating that memory Tfh cells are a major
component of human T cell memory. Memory Tfh cells preferen-
tially become Tfh cells and GC Tfh cells upon reactivation (Hale
et al., 2013)(Figure 2). In humans, memory Tfh cells are hetero-
geneous in phenotype, at least in blood (Schmitt and Ueno,
2013), including a significant fraction of resting memory Tfh cells
that express low amounts of programmed cell death-1 (PD-1).
These PD-1
lo
memory Tfh cells are the most polarized and func-
tional memory Tfh cells, as measured by gene-expression
profiling and B cell help (Locci et al., 2013). As a result, one model
is that those memory Tfh cells are the most likely to retain their
Tfh differentiation program upon reactivation (Figure 2), but this
requires additional studies.
Bcl6 expression is not stable; it requires continuous reinforce-
ment. As such, when a GC Tfh cell leaves a GC and transitions to
a non-GC Tfh cell state (a Tfh cell), Bcl6 expression is reduced
(Kitano et al., 2011; Yusuf et al., 2010). Bcl6 expression is further
reduced as the cell transitions to a fully resting state, becoming a
memory Tfh cell (Choi et al., 2013b; Hale et al., 2013; Liu et al.,
2012). This is not unlike the phenotype of other central memory
CD4
+
T cell subsets, which frequently exhibit relatively low
amounts of canonical master regulator transcription factors.
Interestingly, activated memory B cells induce rapid re-expres-
sion of Bcl6 by memory Tfh cells (Ise et al., 2014), reinforcing
the concept that many features of Tfh cells are highly intertwined
with those of its partners, the B cells.
532 Immunity 41, October 16, 2014 ª2014 Elsevier Inc.
Immunity
Review
The Transcription Factor Network Driving Tfh Cell
Differentiation
Bcl6 is essential for Tfh differentiation and is frequently referred
to as the Tfh master regulator transcription factor. However,
the master regulator transcription factor concept is an oversim-
plification of lymphocyte biology. More than one transcription
factor is critical for any CD4 T cell differentiation program. As
such, the transcription factors Bcl6, RORgt, T-bet, GATA3,
and Foxp3 are now more frequently referred to as ‘‘lineage
defining’’ transcription factors for Tfh, Th17, Th1, Th2, and
pTreg cells, respectively. Even this nomenclature is imperfect,
given the evidence of reversible plasticity by most CD4
+
T cell types (O’Shea and Paul, 2010). It appears that most
cell types can be distinguished by differential expression of a
set of 4–6 transcription factors (TFs) (Ravasi et al., 2010), and
this concept applies to CD4
+
T cell differentiation (Ciofani
et al., 2012). For Tfh cells, several transcription factors are
required in addition to Bcl6. These include Maf, interferon reg-
ulatory factor 4 (IRF4), the activator protein 1 (AP-1) family
member ‘‘basic leucine zipper transcription factor’’ (Batf),
STATs, and E proteins.
IRF4 and Batf are both essential for Tfh cell differentiation
(Bollig et al., 2012; Ise et al., 2011), but they are required for mul-
tiple different CD4
+
T cell programs and can be considered early
T cell activation-associated transcription factors that enable
expression and function of downstream cell-fate-determining
transcription factors (Vahedi et al., 2013). This might occur via
cell-type-specific superenhancer regulation. For example, Batf
is a positive regulator of the Bcl6 gene, and combined expres-
sion of Bcl6 and Maf is required to obtain CXCR5 expression
in vivo in the absence of Batf (Ise et al., 2011). Maf is highly ex-
pressed in Tfh cells and is associated with CXCR5, IL-21, and
IL-4 expression (Bauquet et al., 2009; Hiramatsu et al., 2010;
Ise et al., 2011; Kroenke et al., 2012).
The E protein Ascl2 is a newly recognized contributor to Tfh
differentiation. Constitutive expression of Ascl2 in murine CD4
+
T cells induces CXCR5 expression by some CD4
+
T cells
in vitro (Liu et al., 2014). Ascl2
fl/fl
Cd4
Cre
mice had normal Tfh
cell differentiation and CXCR5 expression, which indicates that
Ascl2 is not a unique regulator of CXCR5 and that there is redun-
dancy among the several E proteins expressed in T cells and
multiple E proteins can enhance CXCR5 expression by binding
enhancer regions (Liu et al., 2014; Miyazaki et al., 2011). The sig-
nals inducing Ascl2 expression or other E-box proteins remain
undefined. Additionally, E protein function is heavily regulated
by Inhibitor of DNA-binding 2 (Id2) and Id3 (Yang et al., 2011a).
Foxp1 and Foxo1 represent transcription factor examples of
the converse biology. Foxp1 and Foxo1 are expressed in resting
naive CD4
+
T cells and are required for quiescence and homing
of naive CD4
+
T cells (Hedrick et al., 2012). Eliminating expres-
sion of either of these two genes—by genetic ablation or, at least
in the case of Foxo1, ubiquitination and degradation by Itch—is
critical for Tfh differentiation (Stone et al., 2013; Wang et al.,
2014; Xiao et al., 2014). These transcription factors appear to
repress Bcl6 and Tfh cell-associated migration genes, among
others, though conflicting Foxo1 data have been reported (Oes-
treich et al., 2012). As such, there are Tfh transcription factors
upstream of Bcl6, as well as transcription factors that coordinate
with Bcl6 or are downstream.
Signal transducers and activators of transcription (STATs) are
also deeply entwined with Tfh differentiation, as they are with all
CD4
+
T cell differentiation pathways (Vahedi et al., 2013). Roles
of STAT proteins in Tfh cells are complex and overlapping.
STAT3 is the most important STAT in murine Tfh differentiation
(Ma et al., 2012a; Ray et al., 2014), while STAT1 and STAT4
can also contribute (Choi et al., 2013a; Nakayamada et al.,
2011; Schmitt et al., 2013). This includes a critical role for
STAT3 in IL-21 expression by murine CD4
+
T cells (Suto et al.,
2008), whereas both STAT4 and STAT3 regulate IL-21 expres-
sion by human CD4
+
T cells (Ma et al., 2009; 2012a; Schmitt
et al., 2009; 2013). Interestingly, STAT1 can either enhance
(Choi et al., 2013a; Nakayamada et al., 2014) or inhibit (Ray
et al., 2014) Bcl6 expression and Tfh cell differentiation.
Regarding murine CXCR5, it is feasible that IL6-driven STAT3
(and STAT1) binds at the CXCR5 promoter in conjunction with
Maf and Batf and drives CXCR5 expression in an E protein-
dependent manner (Ise et al., 2011). This remains to be tested.
In contrast to mice, STAT3 and STAT4 might be equally impor-
tant in human Tfh differentiation (Ma et al., 2012a; Schmitt
et al., 2013)(Figure 1B). In contrast, STAT5 represses Tfh differ-
entiation (Johnston et al., 2012). The opposing roles of STAT3
and STAT5 in Tfh differentiation appear similar to the antago-
nistic roles of STAT3 and STAT5 in Th17 differentiation (Yang
et al., 2011b). These multifaceted regulators of Tfh differentiation
also highlight the need to always consider the complex features
of CD4
+
T cell gene regulation.
The mechanisms by which Bcl6 controls CD4
+
T cells have re-
mained only partially elucidated. Bcl6 is a DNA binding transcrip-
tion factor, and it contains three main domains, the BTB (broad
complex, tramtrack, bic-a-brac), RDII (repressor domain II, or
‘‘middle domain’’), and the Zn finger DNA binding domain
(Figure 3). However, within each of those domains, Bcl6 has
the capacity to interact with a range of proteins, particularly tran-
scription factors and chromatin modifiers (Figure 3). The Bcl6
BTB domain is known to bind multiple other BTB-containing
and non-BTB containing proteins. The Bcl6 Zn finger domain
actually consists of three different Zn finger pairs, one of which
is involved in DNA binding, while the others are involved in pro-
tein-protein interactions (Figure 3). Together, these features
allow for vast combinatorial possibilities for Bcl6 control of
gene expression.
Bcl6 has only been described as a repressor. In GC B cells,
there is no clear evidence of Bcl6 directly binding and activating
any gene, though it causes indirect upregulation of many
genes (Hatzi et al., 2013). In Tfh cells, Bcl6 appears to participate
in control of at least four major categories of genes: cell
migration, repression of alternative fates, Tfh differentiation,
and Tfh products (Figure 4A). Bcl6 expression is closely associ-
ated with CXCR5 expression in vivo (Baumjohann et al., 2011;
Choi et al., 2011; 2013b; Johnston et al., 2009; Pepper et al.,
2011). Nevertheless, CXCR5 expression can be initiated inde-
pendently of Bcl6 (Liu et al., 2012). Furthermore, memory Tfh
cells continue to express CXCR5 without evidence of elevated
Bcl6 expression (Chevalier et al., 2011; Hale et al., 2013; Locci
et al., 2013). Genome-wide Bcl6 occupancy in GC Tfh cells
has revealed that Bcl6 binds over 3,000 genes, with several
key patterns emerging (S.C., unpublished data). Bcl6 binds
many migration-associated genes, apparently inhibiting GC Tfh
Immunity 41, October 16, 2014 ª2014 Elsevier Inc. 533
Immunity
Review
cells from mislocalizing to the T cell zone or sites of inflammation.
Bcl6 expression enhances CXCR5, CXCR4, and PD-1 expres-
sion (Kroenke et al., 2012), but Bcl6 does not appear to regulate
them via direct binding and induction of gene expression (S.C.,
unpublished data). Furthermore, Bcl6 is an important repressor
of alternative cell fates (Figures 4A and 4B), which is discussed
separately in the section below.
Layers of Differentiation
As noted above, the vast majority of GC Tfh cells have highly
conserved gene expression. However, GC Tfh cells interconvert
with Tfh cells; therefore, the GC Tfh cell gene-expression pro-
gram is neither terminal nor immutable. Tfh outside of GCs
have more heterogeneous gene-expression profiles, perhaps
related to their lower expression of Bcl6, resulting in less repres-
sion of alternative cell-fate programs. Heterogeneity among Tfh
cells is not unlike Th17, Th1, Th2, or even Treg cells, where sub-
stantial heterogeneity is observed. For example, it is well estab-
lished that Th17 cells can be converted to Th1 or Treg cells.
IL17
+
IFNg
+
CD4
+
T cells are observed under a number of condi-
tions. There have been longstanding data indicating interconver-
sion of Th1 and Th2 cells. It is quite clear that epigenetic marks
require continuous maintenance and can be changed when cells
enter different environments or experience new external signals
(Mukasa et al., 2010). The behavior of the Tfh differentiation pro-
gram is like that of other CD4
+
T cell differentiation programs in
this regard.
One way to conceptualize this biology is with architectural
blueprints showing layers of differentiation, as if a cell is a multi-
story building and a single floor represents a differentiation pro-
gram (Figure 4B). Each differentiation program has a specific
blueprint, which is the gene-expression program controlled by
a core transcription factor network and external signals. In the
case of the Tfh program, the core transcription factors are
Bcl6, Maf, and STAT3 (STAT3 and STAT4 in humans), with sup-
porting contributions by additional transcription factors. These
transcription factors control four general aspects of Tfh cell
biology, including location, function, and differentiation or posi-
tive reinforcement (Figure 4A). The fourth important attribute of
Tfh biology is repression of alternative cell-fate differentiation
programs. One obvious way this could be accomplished is by
downregulating cytokine receptors necessary for Th1 (IL-12R,
interferon-greceptor-1 [IFNGR1]), Th2 (IL-4R), pTreg (TGF-bR),
or Th17 (IL-23R) cell differentiation. Indeed, Bcl6 directly re-
presses each of these genes (S.C., unpublished data), thereby
cutting off alternative inductive signals. Bcl6 also represses
Blimp-1 (Johnston et al., 2009), which is strongly associated
with non-Tfh cell fates (Figure 4). Nevertheless, external cues
can still induce Th1, Th2, or Th17 differentiation signals in the
cell through the residual cytokine receptor expression. This
can result in expression of some amount of Th1, Th2, or Th17
genes. Thus, Bcl6 inhibits the Th1, Th17, Treg, and Th2 blue-
prints by also directly targeting central Th1, Th17, Treg, and
Th2 transcription factors and cytokine genes for repression
(S.C., unpublished data; Nurieva et al., 2009)(Figure 4B). IFN-
g, IL-17, or IL-5 and IL-13 expression is uncommon in human
GC Tfh cells (Kroenke et al., 2012; Ma et al., 2009), indicating
that the Bcl6 amounts in GC Tfh are sufficient to block Th1,
Th2, and Th17 gene expression in the majority of in vivo
Figure 3. Bcl6 Protein Domain Structure and Interacting Proteins
The functional domains of Bcl6 are shown with regions of interest indicated. Bcl6 interacting proteins are also shown with the Bcl6 regions with which they
interact, if known. Numbers indicate amino acid positions of human Bcl6. Bcl6 is shown in blue. ‘‘BTB’’ is the broad-complex, tramtrack, and bric-a
`-brac domain.
Bcl6 self-dimerizes via the BTB domain, but the self-dimerization is not shown, for simplicity. HDAC, histone deacetylase complex. Ub, ubiquitin. ‘PEST ’indicates
a PEST (proline, glutamic acid, serine, threonine-rich) ubiquitination domain (1 and 2) in Bcl6. ℗symbolizes phosphorylation. Ac symbolizes acetylation, and the
acetylation is mediated by p300. The Zn fingers are numbered 1–6 (e.g., Zn1). NuRD, nucleosome-remodeling and histone deacetylation complex.
534 Immunity 41, October 16, 2014 ª2014 Elsevier Inc.
Immunity
Review
conditions. However, mouse models have demonstrated that,
under conditions of intense polarization, expression of other dif-
ferentiation programs occurs within GC Tfh cells. For example, in
a strongly Th1 systemic chronic lymphocytic choriomeningitis
virus (LCMV) infection GC, Tfh cells express IFN-g(Yusuf et al.,
2010). In the systemic autoimmunity prone BXD2 mice, sponta-
neous GC development is associated with IL-17
+
RORgt
+
GC Tfh
cells (Ding et al., 2013). The canonical Tfh, Th1, Th2, and Th17
blueprints can also interact in unexpected ways, further adding
to the biological complexity, such as enhancement of Tfh cell fre-
quencies in the presence of excessive IFN-g(Lee et al., 2012). In
summary, the majority of GC Tfh cells exhibit a canonical gene
expression profile, which is conserved across species and pre-
dominantly excludes expression of Th1, Th17, Th2, Th9, and
Treg cell-associated genes. Nevertheless, substantial heteroge-
Figure 4. An Architectural Blueprint
Conceptual Model of CD4 T
+
Cell
Differentiation Programs
(A) Regulation of Tfh differentiation by Bcl6 and
cooperating Tfh transcription factors (TFs). Tfh cell
biology can be divided into four categories, indi-
cated in different colors, with representative genes
shown for each category. All differentiation and
product genes shown are upregulated in Tfh cells.
All alternative-fates genes shown are down-
regulated and are grouped in subcategories (Th1,
Th2, IL-2 and Blimp1, Th17, and forkhead box
gene regulation). Genes in the location category
include genes that are upregulated (+) or down-
regulated ().
(B) An architectural blueprint model of Tfh cell
differentiation and how it relates to other CD4
+
T cell differentiation programs, within a single cell.
The Tfh cell differentiation program (shown in 4a) is
projected as a flat plane, as if it were the archi-
tectural blueprint for a floor of a multistory building,
but in this case it is the blueprint within a CD4
+
T cell instead of a building. The Th1 cell program is
projected as the blueprint of another floor. Bcl6
prevents activity of the Th1 cell program in a Tfh
cell by blocking the expression of genes central to
the Th1 cell blueprint. Extracellular signals can
enter as inputs from the surface of the cell, such as
through IL-2R or IFN-gR, shown at the edge of the
Th1 level. Proteins whose genes are inhibited by
Bcl6 are indicated by a red T. The Th17 cell pro-
gram is projected as another blueprint, which is
also inhibited by Bcl6. The Th2 and Treg cell pro-
grams are not shown due to space constraints but
are conceptually analogous to the Th1 and Th17
cell program blueprints.
neity of GC Tfh (and Tfh) phenotypes is
possible depending on environmental
cues.
Complexities of Tfh Cell
Differentiation and Memory
An alternative model to consider is that
Tfh differentiation is a secondary pro-
gram, such that there are Th1 cell-type
Tfh (Tfh1), Th2 cell-type Tfh (Tfh2),
and Th17 cell-type Tfh (Tfh17) cells
(Figure S1)(Crotty, 2011). In this concep-
tual framework, there are Th1 effector
cells and Tfh1 cells in response to a viral
infection, for example. In support of this model, Tfh cells in the
context of viral infection express some T-bet and IFN-g(John-
ston et al., 2009; Yusuf et al., 2010), and there are rare IFN-g
+
Tfh in L. major-infected mice (Reinhardt et al., 2009). There are
also examples of IL-17
+
Tfh cells in autoimmune prone BXD2
mice (Ding et al., 2013). IL-5 or IL-13 Th2 cytokine-expressing
Tfh cells were reported (Zaretsky et al., 2009). However, a
more recent extensive study concluded that Tfh cells do not ex-
press IL-13 or IL-5 (Liang et al., 2012). Transfer experiments are
challenging to interpret, since the outcome can be the result of
outgrowth of a small number of cells that did not share the
biology of the majority of the cells. Cell transfers appear to
show that Th2 cells can convert into Tfh cells (Zaretsky et al.,
2009), and Th17 cells can convert into Tfh cells (Hirota et al.,
2013). However, in other studies, Tfh, Th1, and Th17 cells have
Immunity 41, October 16, 2014 ª2014 Elsevier Inc. 535
Immunity
Review
largely retained their identities after transfer (Choi et al., 2013b;
Hale et al., 2013; Weber et al., 2012). When faced with the human
biology, the interpretations are more complicated. While very
few GC Tfh cells express non-Tfh cell cytokines in lymphoid
tissue (Kroenke et al., 2012; Ma et al., 2009), when observing
memory Tfh cells in human blood a high percentage of the mem-
ory Tfh cells express chemokine receptors generally affiliated
with human Th1, Th17, or Th2 cell memory cells (Morita et al.,
2011). However, conventional chemokine receptor-based defini-
tions of human blood central memory Th17 and Th2 cells are
flawed, as only a small percentage of those cells actually pro-
duce Th17 or Th2 cell-associated cytokines upon restimulation.
Additional complexities to understanding CD4
+
T cell differentia-
tion programs include overlapping biology, including the obser-
vation that Tbet can form a complex with Bcl6 and both inhibit
major functions for Bcl6 and enhance other functions (Oestreich
et al., 2012). Finally, there are a great deal of data not in support
of the secondary program conceptualization of Tfh cell differen-
tiation, many of which were stated earlier in this review. In partic-
ular, Tfh cell differentiation can start at DC priming and can be
distinguished from Th1 cells within the first two cell divisions
in vivo during an acute infection (Choi et al., 2011). Second,
Tfh cell differentiation is independent in that it does not require
Th1, Th2, or Th17 cell programming (Nurieva et al., 2008).
Both conceptual models do have strengths. Although it is
appealing to have a simple shared nomenclature, this is inher-
ently problematic for cells as heterogeneous as CD4
+
T cells
are. From a semantic perspective, this is in part a question of
the perceived primary biology of the cell in question. Is it primarily
a Tfh cell, helping B cells? Or is it primarily a Th1 cell, inducing
inflammation in an infected tissue? Treg cells are an illustrative
example here. While there are clearly Tbet
+
or RORgt
+
Tregs
(Th1-related and Th17-related), these cells are clearly still Tregs,
because their primary biology is to dampen and regulate immune
responses. A similar perspective is applicable to Tfh cells. While
there can be Tbet
+
or RORgt
+
Tfh cells, these cells are clearly still
Tfh cells, because their primary biology is to help B cells. Hence,
the conceptual frameworks discussed thoroughly earlier in the
review and shown in Figures 1B and 4B seem most relevant.
Th1, Th2, or Th17 cell-associated cytokines produced by Tfh
cells or GC Tfh cells can be instructive for class-switch recombi-
nation and are therefore not irrelevant. However, it must be noted
that the importance of this cytokine biology appears to be exac-
erbated in mice, because the difference in immunoglobulin G
(IgG) class switch recombination between Th1 and Th2 condi-
tions is dramatic in mice (murine IgG1 and murine IgG2a/b/c
have very different functions), whereas human IgG1 (the domi-
nant isotype) has broad functionality. In addition, while IFN-gis
a major class switch recombination factor in mice, its role in
human IgG class switching appears to be trivial (Holland and
Casanova, 2006). In summary, Tfh cells can exhibit features
consistent with the Tfh1/2/17 cell conceptual framework, but
on balance the majority of the data support Tfh cells as a distinct
cell type as the more instructive conceptual framework.
Within the context of categorizing cells and differentiation pro-
grams, T follicular regulatory (Tfr) cells are a separate issue,
because these cells are thymic Treg (tTreg or ‘‘natural Treg’’)
cells that also express Bcl6 and CXCR5 (Chung et al., 2011; Lin-
terman et al., 2011; Sage et al., 2013; Wollenberg et al., 2011).
They are analogous to type-specific Treg cells, such as Th1
cell-specific Treg cells, which express Tbet as a mechanism to
express CXCR3. Expression of the appropriate chemokine re-
ceptor is a necessary part of Treg cell biology, so that they
migrate to the same location of the effector cells (Tfh or Th1 cells
in these examples) such that they can dampen immune re-
sponses at that site. Bcl6
+
CXCR5
+
induced pTreg cells have
yet to be identified.
Bcl6 does have some role in the development of T cell mem-
ory, which also impacts interpretations of Tfh cell biology at
different time points. While Bcl6 has a role in development of
memory CD8 T cells, the cell intrinsic effect of Bcl6 deficiency
is modest (Cui et al., 2011). In CD4
+
T cells, the role of Bcl6 in
memory requires further investigation. Early experiments with
Bcl6-deficient CD4
+
T cells lacked key controls for cellular rejec-
tion (Ichii et al., 2007), whereas more recent studies did not
examine Bcl6-deficient CD4
+
T cells at memory time points
(Hale et al., 2013; He et al., 2013; Pepper et al., 2011). An addi-
tional challenge to studying Tfh cell memory is that active Tfh
cell responses continue for much longer than Th1 or Th2 cell re-
sponses after an acute immunization or infection. Tfh cells are
active for the duration of the GCs, with continuous exposure to
antigens, which frequently last for 60 or more days. Therefore,
to stringently study resting Tfh cell memory, it is likely necessary
to wait more than 90 days, depending on the model system,
while resting Th1 or Th2 cell memory develops by day 30 after
an acute antigen exposure.
Tfh Cell Function
The most prominent role of Tfh cells is their requirement for GC
development and function. The GC is the primary site of B cell
affinity maturation. The extraordinary process of Ig gene somatic
hypermutation and selection is one of the miracles of immu-
nology. A GC reaction is effectively evolution in miniature. Regu-
lation of Tfh cell help is central for achieving the goal of GC
responses, which is to generate and select GC B cells with higher
affinity for the pathogen (Victora and Nussenzweig, 2012). In
GCs, B cells circulate through two regions: the light zone (LZ)
and the dark zone (DZ). In the LZ, GC B cells bind to antigen
and present antigen peptide:MHC complexes to Tfh cells that,
in turn, provide help signals to GC B cells that are essential for
their survival and proliferation. GC B cells that receive survival
signals and then migrate to the DZ, where they undergo prolifer-
ation and somatic hypermutation (SHM), allowing for the gener-
ation of BCRs with a spectrum of affinities to antigen. These
mutated GC B cells then move back to the LZ, where the highest
affinity B cells are selected again by the Tfh cells for another
round of proliferation and mutation. Multiple studies have re-
vealed the roles of Tfh cells in regulating GCs (Crotty, 2011; Vic-
tora and Nussenzweig, 2012). Tfh cells regulate GC size (Hams
et al., 2011; Johnston et al., 2009; Rolf et al., 2010), restrict
low-affinity B cell entry into the GC, support high affinity B cell
occupancy of the GC (Schwickert et al., 2011), and select
high-affinity B cells during affinity maturation (Good-Jacobson
et al., 2010; Victora et al., 2010). In addition, most GC B cells
cannot trigger BCR signaling (Khalil et al., 2012). Therefore, the
GC B cells are dependent on help signals from the Tfh cells to
discriminate which GC B cells proliferate. Tfh cells selectively
provide help to the B cells with the most antigen peptides, which
are the high-affinity B cells that bound and endocytosed the
536 Immunity 41, October 16, 2014 ª2014 Elsevier Inc.
Immunity
Review
most antigen. Surprisingly, the amount of help provided by the
Tfh cell directly translates to the number of cell divisions—and
mutations—a GC B cell will undergo in the DZ in a single selec-
tion cycle (Gitlin et al., 2014). Therefore, Tfh cells regulate the
mutation diversity of BCRs in a GC. Strong Tfh help to B cells
can result in GC B cell accumulation of multiple mutations in a
single round of selection, which may be important for generating
rare multiple mutation combinations, particularly if a single muta-
tion happens to be deleterious on its own. For example, develop-
ment of broadly neutralizing antibodies against HIV is very diffi-
cult and requires extensive somatic hypermutation (Burton
et al., 2012). Some of these antibodies develop new disulfide
bonds that stabilize long CDR loops (Doria-Rose et al., 2014).
Single cysteines are likely to be deleterious, and are unlikely to
be positively selected. Simultaneous generation of two cysteine
mutations in a single selection cycle might be an example of how
strong Tfh cell help to GC B cells controls the mutation spectrum
of GC B cells and might allow some B cells to overcome large
hurdles in the evolutionarily landscape to develop high affinity
for their target antigen.
The help signals provided by Tfh cells to GC B cells consist of
both cytokines and cell-surface receptors. While the help signals
are incompletely characterized, CD40L, IL-21, and IL-4 are major
‘‘help’’ molecules produced by GC Tfh cells to keep GC B cells
alive and induce their proliferation (Crotty, 2011). IL-21 and IL-
4 are also potent inducers of IgG1 class switch recombination
for human B cells (Avery et al., 2008), and IgG1 is the most prev-
alent class-switched immunoglobulin. The help factors IL-21 and
IL-4 are not simply produced by Tfh cells upon TCR engage-
ment; they are regulated by additional cell-surface molecules
that effectively communicate additional dialog between the Tfh
cell (the provider of help) and the GC B cell soliciting help. IL-4
is produced by GC Tfh cells in a SLAM-dependent manner (Yu-
suf et al., 2010), and SLAM is selectively upregulated on LZ GC B
cells (Victora et al., 2012). Separately, ICOS triggering is impor-
tant for Tfh cell production of IL-21 (Morita et al., 2011). Manipu-
lating the amount of Tfh cell help given to B cells dramatically
alters affinity maturation. Indiscriminate Tfh cell help to both
high and low affinity B cells leads to the generation of lower
affinity antibodies over time (Victora et al., 2010). Therefore,
the outcome of the GC B cell response depends on the proper
regulation of Tfh cell help to high affinity versus low affinity B
cells. For this reason, negative signaling through SLAMF6 might
serve a critical purpose in limiting Tfh cell help (Kageyama et al.,
2012), thereby enhancing selective pressure.
Inhibitory signals to Tfh cells might limit Tfh cell help signals or
Tfh cell proliferation or both. One of the central challenges of Tfh
cells in GCs is that they are constantly exposed to antigen and
must retain sensitive TCR signaling—so as to distinguish be-
tween GC B cells with modest differences in their numbers of
p:MHC complexes—but the Tfh cell needs to not respond by
proliferating, in contrast to most effector CD4
+
T cells. Instead,
the Tfh cell needs to respond to that TCR signaling by only
providing transient help to the cognate B cells. After all, the pur-
pose of the GC is for the GC B cells to rapidly proliferate, mutate,
and evolve, not the Tfh cell. As such, control of multiple compo-
nents of TCR signaling and downstream pathways (including
regulation of transcription elongation by Cyclin T1 [Chen et al.,
2014]) might be essential for striking the balance in Tfh cells
between sensitive sensing of p:MHC and unwanted proliferation.
The extremely high amount of PD-1 expressed by GC Tfh cells
critically contributes to limiting GC Tfh cell proliferation in GCs
by dampening TCR signaling. Nevertheless, excessive availabil-
ity of the PD-1 ligand PD-L1 on GC B cells can occur and is asso-
ciated with defective Tfh cell function (Cubas et al., 2013). High
amounts of PD-L1 on GC B cells results in reduced ICOS and
IL-21 expression, resulting in minimal help to the PD-L1
hi
GC B
cells (Cubas et al., 2013). In addition to PD-1 and SLAMF6, Tfh
cells express multiple additional inhibitory receptors, which
might control both Tfh cell proliferation and function. Immuno-
therapy targeting Tfh cells by blocking PD-L1 and LAG-3 in a
mouse malaria model led to an increase in Tfh cell and GC B
cell numbers, followed by rapid development of protective anti-
bodies and clearance of the Plasmodium (Butler et al., 2012). In
summary, Tfh cell help to GC B cells is provided via a combina-
tion of secreted and surface bound help molecules, which are
counterregulated by inhibitory molecules that can critically affect
the amount of help provided, the duration of the T:B interaction
(which is related to the amount of help provided), or the cocktail
of molecules expressed by the Tfh cell.
Negative regulation of the GC is also accomplished by Tfr
cells, introduced earlier in this review. Tfr cells can have potent
inhibitory roles in GCs, and it is thought that the ratio between
the Tfh and Tfr cells is an important determinant of the GC reac-
tion. Nevertheless, there are many unanswered questions about
Tfr cells. It is unclear whether the Tfr cells primarily function by
interacting with T cells (Tfh cells in this case), which is the primary
mechanism by which tTregs cells act, or alternatively, the Tfr
cells might mainly act directly on the GC B cells. There is
currently uncertainty whether Tfr cells can regulate immune re-
sponses in an antigen-specific manner, and it might be that their
primary role is to eliminate autoreactive B cells that arise via mu-
tation in the GC (Linterman et al., 2011).
It is worth noting that while the most important and well-stud-
ied role of Tfh cells is their requirement in GCs, Tfh cells also have
critical roles outside the GC. This is an area that is understudied.
In addition, there are GC-independent memory B cells, which
form early during responses and generally have no mutations.
These memory B cells are Tfh cell-independent (Kaji et al., 2012).
Tfh Cells in Disease
Tfh cells are essential for the generation of most isotype
switched and affinity matured antibodies, and therefore they
have an obvious role in protective immunity against pathogens.
Antibodies are necessary for the control of LCMV infection,
and defects in Tfh cell frequencies result in failure to control
LCMV (Fahey et al., 2011; Harker et al., 2011). LCMV has a sur-
face that is very difficult for antibodies to bind, hence substantial
T cell help to B cells is necessary to drive the slow generation of B
cell responses capable of controlling the viremia. A similar prob-
lem exists for HIV, made even worse by the extreme mutability of
HIV. Tfh cell frequencies are associated with the amount and
quality of antibody responses against SIV (simian immunodefi-
ciency virus, a close relative to HIV) in SIV-infected macaques
(Petrovas et al., 2012). Impaired Tfh cell help to B cells is
observed in HIV-infected individuals, which appears to exacer-
bate the difficulty in generating neutralizing antibodies against
HIV (Cubas et al., 2013). Conversely, individuals who have un-
usually elevated frequencies of highly functional memory Tfh
Immunity 41, October 16, 2014 ª2014 Elsevier Inc. 537
Immunity
Review
cells (PD1
lo
CXCR3
CXCR5
+
) are more likely to make broadly
neutralizing antibodies against HIV (Locci et al., 2013), again
consistent with the hypothesis that Tfh cells are a key limiting
resource for the development of high-affinity B cell responses.
Almost all licensed human vaccines work on the basis of long-
term protective antibody responses, and therefore it is reason-
able to assume that Tfh cells are mediators of the development
of the protective immunity generated by licensed human vac-
cines. There is evidence that Tfh cell help is a limiting factor in
humans for generating antibody responses after immunizations
(Bentebibel et al., 2013; Duan et al., 2014; Pallikkuth et al.,
2012; Schmitt et al., 2013). Therefore, learning to control Tfh
cells would almost certainly enhance development of new or
improved vaccines. As described above, boosting Tfh cell
numbers with immunotherapy led to dramatically improved anti-
body responses in mouse malaria (Butler et al., 2012). Therefore,
it might also be that vaccines that employ monoclonal antibodies
targeting Tfh cell inhibitory pathways could adjuvant vaccine
efficacy.
Tfh cells are not only important in control of pathogens, they
are also important in control of the commensal microbiota. The
predominant antibody isotype at mucosal surfaces is IgA, and
the majority of IgA is T-dependent, based on data from MHCII-
or T cell-deficient animals. In addition, in mice that had normal
IgA levels but a defect in productive GC somatic hypermutation,
the microbiota was expanded and increased susceptibility to
the intestinal pathogen Yersinia pestis was observed (Wei
et al., 2011). Thus, Tfh cells are required for sufficient antibody
response quality to control both commensals and pathogens.
Nevertheless, intestinal immune responses are characterized in
many ways by their balance between protecting against mi-
crobes and avoiding undo inflammation. This paradigm has
been seen to also hold true for Tfh cells, as a defect in Tfr cells
can result in more Tfh cells, larger antibody responses, and
less microbiota diversity, which, paradoxically, can result in
less healthy gut homeostasis (Kawamoto et al., 2014). Interest-
ingly, Tfh cells are also essential for IgE production (Liang
et al., 2012), and therefore Tfh cells are important in allergic
responses; however, the literature on Tfh cells in allergy is
currently limited and this is an area in need of much more
investigation.
Tfh cells are central players in a number of autoimmune dis-
eases, and it is hoped that a greater understanding of Tfh cells
can result in new therapeutic approaches against major autoim-
mune diseases. Increased frequencies of Tfh-like cells (CXCR5
+
and PD-1
hi
or ICOS
hi
) in peripheral blood are observed in subsets
of patients with Sjogren’s syndrome (Simpson et al., 2010;
Szabo et al., 2013), juvenile dermatomyositis (Morita et al.,
2011), and systemic lupus erythematosus (He et al., 2013; Simp-
son et al., 2010). Each of those diseases is associated with
extensive autoantibody production. Autoimmune diseases for
which autoantibodies play a direct pathogenic role are likely to
have Tfh cells as an important component of the disease, for
example, granulomatosis with polyangiitis (GPA). Separately,
there are functions of Tfh cells that might be unrelated to anti-
body responses, per se. For autoimmune diseases like SLE, au-
toantibodies are considered primarily markers of disease, not
causes of disease. Interestingly, a common variation of SLE in
humans is lupus nephritis, which can result in severe kidney
dysfunction associated with inflammation and the accumulation
of high concentrations of autoantibodies. Importantly, ectopic
clusters of Tfh cells and B cells can be found in the inflamed kid-
neys of such patients, including GC Tfh cells and GC B cells (Liar-
ski et al., 2014). These and other data suggest that Tfh cells
might be major regulators of ectopic follicles in autoimmune
diseases (Craft, 2012). Thus, Tfh cells might contribute to auto-
immune diseases both by facilitating the aberrant generation of
autoantibodies and by facilitating the formation or maintenance
of ectopic follicles, which serve as nucleation points for other
cells that might be directly pathogenic in the autoimmune
disease.
Although roles for Tfh cells in infectious diseases, allergy, and
autoimmunity were expected, it was not anticipated that Tfh cells
would be relevant for cancer immunity. Therefore, it was surpris-
ing that a strong positive correlation was observed in breast
cancer between a Tfh gene signature in the tumor tissue and
long-term patient survival (Gu-Trantien et al., 2013). Histology
confirmed that a Tfh cell gene signature was associated with
infiltration of the tumor margin with Tfh cells and the develop-
ment of B cell follicles or ectopic lymphoid organ-like structures.
More surprising, similar results were found for human colorectal
cancer (Bindea et al., 2013). Positive outcomes in controlling the
cancers were associated with Tfh cell gene signatures, including
CXCL13 and IL-21 expression. Even more intriguing, tumor dele-
tion of the CXCL13 gene was associated with cancer progres-
sion (Bindea et al., 2013). Therefore, this invites the speculation
that Tfh cells might have shared roles in cancer immunity and
autoimmune diseases independently of helping antibody re-
sponses. The Tfh cells might facilitate or maintain ectopic B
cell-rich lymphoid structures, sustaining a local microenviron-
ment that is nurturing for other immunological cell types with
more direct roles in affecting disease progression or regression,
whether they be CTL in the context to tumor immunity (Bindea
et al., 2013; Gu-Trantien et al., 2013) or Th17 cells in the context
of multiple sclerosis (Hauser et al., 2008).
In conclusion, much has been recently discovered about the
biology of Tfh cells and germinal centers. While major knowledge
gaps remain, and Tfh cell biology is clearly complex, it is never-
theless easy to predict that in the coming years we will see an
ever-growing impact of the study of Tfh cells as we appreciate
more and more that Tfh cells have pivotal roles in a range of
diseases.
SUPPLEMENTAL INFORMATION
Supplemental Information includes one figure and three movies and can
be found with this article online at http://dx.doi.org/10.1016/j.immuni.2014.
10.004.
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