2.1. Characterization of T Follicular Helper Cells
The presence of Tfh cells within the GC reaction ensures the survival and growth/expansion of antigen-specific B cells during the process of affinity maturation [
7,
8,
9]. In the absence of T cells, GC form, but are not maintained [
47,
48]. The ability of activated CD4
+ T cells to migrate from the T cell zone into the B cell follicle is mediated by the downregulation of C-C chemokine receptor type 7 (CCR7) following TCR ligation that reduces tethering of T cells to the T cell zone where the ligands for CCR7 (the chemokines (C-C motif) ligand 19 (CCL19/ELC) and (C-C motif) ligand 21 (CCL21)) are expressed [
10,
49]. The reduction of CCR7 expression occurs in conjunction with increased expression of C-X-C chemokine receptor type 5 (CXCR5) [
50,
51]. CXCR5 is constitutively expressed on B cells where it is important for the formation of B cell follicles [
52]. On CD4
+ T cells, CXCR5 is expressed transiently upon TCR ligation [
10] and is maintained at high levels on Tfh cells [
7,
8]. CXCR5
+CCR7
−CD4
+ T cells migrate toward the ligand for CXCR5, namely chemokine (C-X-C Motif) Ligand 13 (CXCL13), which is produced by FDCs that mark the anatomical site where the GC reaction forms [
49,
50,
51,
53].
Apart from their location in the B cell follicle, Tfh cells are distinguished from other T helper cell subsets by the elevated expression of molecules that facilitate T and B cell collaboration. Direct interaction of CD40 on GC B cells and CD40L expressed by Tfh cells is indispensable for GC formation and plasma cell differentiation [
54,
55,
56,
57]. Tfh cells characteristically express high levels of ICOS, programmed cell death 1 (PD-1), the transcriptional repressor B cell lymphoma 6 (Bcl-6) as well as cytokines that influence B cell differentiation and antibody production, such as interleukin (IL)-21, IL-4 and interferon-γ (IFN-γ) [
24] (
Figure 1 and
Table 1). In contrast to murine Tfh cells, human Tfh cells have been found to also express the cytokine IL-10, which has important functions in human B cell differentiation [
58]. The engagement of ICOS with its ligand (ICOS-L) expressed on antigen presenting cells (APCs) such as B cells, leads to the production of helper cytokines such as IL-4, IL-10 [
11,
59] and IL-21 [
13]. The Tfh cell produced cytokine IL-21 [
13,
18] has been shown to be a potent inducer of plasma cell differentiation in both mice and humans
in vitro [
17,
60,
61] and the importance of IL-21 for GC B cells is also well established [
15,
16].
Figure 1.
Cytokines in the germinal center (GC) reaction. Schematic diagram showing the cytokines that are important for the GC reaction and the action of these cytokines on different GC cell subsets. The relative importance of any given cytokine depends on the type of immune response during which it is expressed. Interleukin (IL), interferon-gamma (IFN-γ). *IL-17 production by T helper cells happens in dysregulated GCs during autoimmunity.
Figure 1.
Cytokines in the germinal center (GC) reaction. Schematic diagram showing the cytokines that are important for the GC reaction and the action of these cytokines on different GC cell subsets. The relative importance of any given cytokine depends on the type of immune response during which it is expressed. Interleukin (IL), interferon-gamma (IFN-γ). *IL-17 production by T helper cells happens in dysregulated GCs during autoimmunity.
Microarray analyses of the Tfh cell transcriptome from both mice and humans revealed a unique gene expression profile that distinguished Tfh cells from other T helper cell subsets [
18,
58,
62,
63]. Tfh cells were observed to express the highest amounts of IL-21 as well as the intracellular adaptor protein SAP (SLAM-associating protein) and the transcription factor Bcl-6 [
18,
62,
63]. Studies using the Roquin mouse model have shown a remarkably similar transcription profile in mouse and human Tfh cells, with the most highly expressed transcripts in Tfh cells (such as
Il21,
Cxcr5,
CD84,
Cxcl13,
Bcl6 and
Pdcd1, which encodes PD-1) detected in both organisms [
62].
As the study of Tfh cells has progressed, the term Tfh cells has been used to describe CD4
+ T cells that express CXCR5, indicating their B cell homing potential rather than localization to the GC or ability to support an affinity matured antibody response. By this definition, CXCR5hi PD1hi CD4
+ T cells have been detected in the blood in humans and mice [
7,
8,
64,
65]. Whilst the origin of these cells remains unknown, CXCR5
+ CD4
+ memory T cells have been observed to migrate into the B cell follicle in response to secondary antigen challenge indicating that the maintenance of CXCR5 expression on memory CD4
+ T cells can support immunity [
66,
67,
68].
2.2. T Follicular Helper Cell Differentiation
Studies collectively demonstrate that Tfh cell differentiation is a multistage process with key checkpoints regulating the formation, migration, expansion and survival of this T helper cell subset [
24]. Upon recognition of peptide-MHC class II presented by dendritic cells (DCs) in the T cell zone, CD4
+ T cells lose expression of CCR7 and upregulate CXCR5 in a Bcl-6 dependent manner [
7,
8,
69,
70]. The activated GC Tfh “precursors” interact with cognate B cells at the T-B border, and Ag-primed T helper cells with the highest affinity for antigen [
71] are thought to maintain CXCR5 expression [
50,
51,
72]. During this “second round” of cognate interaction, the primed CD4
+ helper cells upregulate Bcl-6 expression and become fully differentiated Tfh cells [
69,
70,
73].
The differentiation, expansion and survival of Tfh cells are dependent upon signals from both DCs and B cells. Like other CD4
+ T cell subsets, activation of Tfh cell “precursors” requires interaction with dendritic cells expressing peptide antigen in the context of MHC class II molecules. Detailed analyses of Tfh cell development revealed a broad upregulation of CXCR5, ICOS, Bcl-6, PD-1 and GL7 on CD4
+ T cells following early (day 2–3) interaction with dendritic cells (DC) [
74,
75,
76]. As higher TCR affinity has been associated with a preference for Tfh cell differentiation [
71], prolonged interactions with DCs during the first 24h of priming leading to extended TCR and costimulatory receptor engagement as well as cytokine exposure, may bolster the Tfh differentiation program [
77,
78,
79]. Tfh cells are able to develop in the absence of B cells, provided that adequate stimulation is available to the T cells in the form of peptide antigen-MHCII complexes on other APCs [
79]. This finding may reflect the ability of B cells to act as a sufficient source of antigen for Tfh cells, but questioned whether B cells also provide any unique signals.
Although B cells are dispensable during the priming phase of CD4
+ T cells, as well as in the initial steps of Tfh cell differentiation, they are of crucial importance for the maintenance and function of Tfh cells during the GC reaction. B cells provide an important function in supporting the expansion/survival of CD4
+ T cells [
80]. Notably, B cells support the maintenance of the Tfh cell phenotype as Bcl-6 expression and Tfh cell commitment is interrupted in the absence of T–B cell interactions [
74]. Interaction with antigen-presenting B cells on the T-B border leads to the SAP-mediated secondary upregulation of Bcl-6 stabilizing the expression of CXCR5 on CD4
+ T cells, who are then able to migrate into the GC and fully differentiate into Tfh cells [
81,
82,
83]. Signals from ICOSL expressed on activated B cells promote persistent Tfh cell motility thus aiding Tfh migration in the B cell follicle [
84].
Tfh cells differentiate under different contexts and thus must be able to assimilate a variety of input signals and this is reflected in the contribution of several transcription factors that are important for Tfh cell differentiation. T cell intrinsic Bcl-6 activity is required for Tfh cell development and GC responses to T-dependent antigen [
69,
70,
85]. Bcl-6 can inhibit the differentiation of other (non-Tfh) CD4
+ T cell subsets and has been shown to antagonize transcription factors important for Th1, Th2 and Th17 differentiation [
69,
70]. Both IL-6 and IL-21 have been shown to upregulate the expression of B
cl-6 [
14,
86]. Critical molecules for T–B cell interactions such as SAP, CD40L, PD-1, ICOS and CXCL13 are induced by the Tfh cell master transcription factor Bcl-6 [
87]. Whilst Bcl-6 expression is high in Tfh cells, the Bcl-6 antagonist Blimp-1 is most highly expressed by other CD4
+ T cell subsets [
85,
88,
89]. Another transcription factor which has been shown to be important for optimal Tfh cell numbers is c-Maf, which induces IL-21 production in a ICOS-dependent manner [
90] by directly binding the IL-21 promoter [
91]. In addition, mice deficient in the transcription factor IRF4 (
Irf4−/−) are unable to upregulate Bcl-6, CXCR5 and ICOS due to an T cell intrinsic defect and thereby fail to generate Tfh cells [
92,
93]. IRF4 contributes to Tfh cell differentiation, but this role is not Tfh cell specific, since
Irf4−/− mice also exhibit defects in the differentiation of Th2, Th9 and Th17 cell subsets [
94,
95,
96]. Furthermore, an CD4
+ T cell intrinsic dependency on NF-κB-signaling for Th2 and Tfh cell induction in response to T-cell dependent Ag was found in adoptive transfer experiments with
NF-κB1−/− CD4
+ T cells [
97]. NF-κB1 deficiency was shown to have detrimental effects on the production of Th2 cytokines IL-4 and IL-13 and even more notably suppressed the induction of CXCR5, whereas expression of other Tfh cell markers such as
Bcl-6, IL-21, CXCR4, OX40 mRNA as well as PD-1 protein was not impaired [
97]. The importance of signaling through the trans-membrane receptor Notch for Tfh differentiation as well as IL-4 secretion by CD4
+ T cells has been shown in mice with a CD4
+ T cell specific deletion in both Notch1 and Notch2 (
N1N2−/−) [
98]. Notch-signaling is suggested to influence Tfh cell differentiation through its impact on the balance of
Bcl6 and
Blimp1 mRNA expression [
98].
Although Bcl-6 is critical for the differentiation of Tfh cells, it is not exclusively expressed in Tfh cells. More recently, a Tfh specific transcription factor has been identified that drives the differentiation program of Tfh cells. Achaete-scute homologue 2 (Ascl2)—a basic helix–loop–helix (bHLH) transcription factor was selectively upregulated in Tfh cells. Ectopic expression of
Ascl2 upregulated CXCR5 (but not Bcl-6) and downregulated CCR7 expression in T cells
in vitro, as well as both accelerating the migration of T cells to the follicles and Tfh cell development in mice [
99].
2.3. T Follicular Regulatory Cells
Regulatory T cells (Tregs) that could suppress the humoral immune response [
1,
100] and CD25
+ T helper cells that localize to the GC [
101,
102] have been described over the past four decades. However, it was more recently that a population of Tregs with follicular homing ability was located within the GC. CXCR5hi PD1hi FoxP3
+ Tregs within the GC were named T follicular regulatory (Tfr) cells [
31,
103,
104]. Foxp3
+ Tfr cells originate from natural (thymus-derived) Treg cells (nTregs) and acquire features of Tfh cells, such as expression of CXCR5 [
10,
31] and high expression of PD-1 [
65]; however, unlike Tfh cells, they lack expression of CD40L, IL-4 and IL-21 [
31,
103,
104].
Tfr cells also possess an activated Treg phenotype, expressing high levels of GITR, CTLA-4 and IL-10 [
31,
103] (
Table 1) and the transcription factor Helios, reported to be expressed by thymic nTregs but not by “inducible” Tregs (iTregs), which are induced to express FoxP3 following antigen stimulation in the periphery from FoxP3
− CD4
+ T cells [
105,
106]. Adoptive transfer of FoxP3
+ Treg cells into congenic recipient mice demonstrated that Tfr cells originate from nTregs and not from extrathymic CD4
+ T cell populations such as (FoxP
−) Tfh cells [
31,
103]. Tfr cells exhibit a negative regulatory function on both Tfh cells and GC B cells, as well as suppressing antigen-specific antibody production [
31,
107,
108]. By contrast, a recent study showed that Tfr cells reduce the levels of IL-2 by utilizing IL-2 within the GC [
109]. Since IL-2 inhibits Tfh cells [
110], the utilization of IL-2 by Tfr cells may have a positive regulating effect on the GC reaction. Taken together, these findings suggest that Tfr cells may operate to balance the magnitude of the GC output, but aspects of their function remain incompletely understood.