T follicular helper (Tfh) cells were discovered just over a decade ago as germinal centre T cells that help B cells make antibodies. Included in this role is affinity maturation and isotype switching. It is here that their functions become less clear. Tfh cells principally produce IL-21 which inhibits class switching to IgE. Recent studies have questioned whether the germinal centre is the main site of IgE class switching or IgE affinity maturation. In this review, I will examine the evidence that these cells are responsible for regulating IgE class switching and the relationship between Tfh cells and T helper 2 (Th2) effector cells.
Common allergic diseases involve both humoral (IgE) and cell-mediated T helper 2 (Th2) mechanisms that drive inflammation. In addition, Th2 cells also regulate isotype switching to IgE. This is certainly true in vitro and the essential signals shown to be CD40L and IL-4 and to a lesser extent IL-13. At the beginning of the twenty-first century, several groups reported that a chemokine homing receptor CXCR5 was expressed on a unique population of T cells with specialized functions for switching B cells and affinity maturation. These cells were called T follicular helper (Tfh) cells and they were found in germinal centres (Figure 1).
Th1 and Th2
The observation by Mosmann and colleagues that CD4 T cells could be subdivided according to their cytokine profile1 and function2 dramatically changed our understanding of what T cells did. Mosmann showed that Th1 CD4 T cells could enter peripheral tissues and recruit macrophages and neutrophils, while Th2 cells in the lung, for example, recruited eosinophils and drove mucus hypersecretion and airway hyper-responsiveness. What was not clear was how these cells could mediate both sets of functions. Expression of CCR7 and CD6L helps explain how central memory T cells are recruited to lymph nodes and CCR2 has been shown to facilitate effector memory T cells' entry to the lung, gut and other tissues. However, the distinction between CD4 T cells that help B cells and those that participate in cell mediated immunity is less clear. This has been a cause of considerable confusion to students who would try to separate these functions based on the Th1/Th2 paradigm.
What are Tfh cells
A series of papers at the beginning of the twenty-first century identified a subset of germinal centre CD4 T cells that differed from the T cells described above.3,4,5 These were first termed T follicular helper (Tfh) cells by Chtanova et al.6 Germinal centre localized Tfh cells expressed the chemokine receptor CXCR5 as well as costimulatory molecules CD40L7 and ICOS.8 IL-21 is required for germinal centre formation for T-cell dependent antigens.9 However, Tfh CD4 T cells can secrete both IL-4 and IL-1010 as well as IL-21.6 Recently, Tfhs cells have been shown to make IL-4 but not IL-13, while tissue Th2 cells made both IL-4 and IL-13.11 Furthermore, Tfh cells that made IL-4 expressed low levels of GATA-3, whereas Th2 cells expressed high levels of GATA-3. Another transcription factor that may drive IL-4 in Th2 cells is conserved non-coding sequence 2.12 conserved non-coding sequence 2-active cells have been found in germinal centres and expressed CXCR5 and BcL613 as well as IL-21 and IL-4. One transcription factor that directs Tfh differentiation is Bcl-6.14 Thus, Tfh and Th2 cells show clear differences, although it is unclear how these arise.
What is the allergic immune response?
Allergic disease is driven by both IgE and Th2 inflammatory processes that synergize. Why some organs are more or less affected is unclear, but there are tissue specific susceptibilities. Models in rats15,16,17 and subsequently in mice18 have shown that Th2 cells alone can drive many of the pathological processes seen in asthma including eosinophil infiltration, IL-4,19 IL-520 and IL-13 that causes mucus hypersecretion and airway hyper-responsiveness.21
How is IgE class switching controlled?
Immunoglobulin class switching by B cells is dependent on two signals. The first is delivered by specific cytokines that activate the promoter of the relevant immunoglobulin isotype22 and the second by CD40L expressed on the CD4 T cells.23 IL-4 was shown24 to switch B cells to IgE and subsequently interferon-γ was identified as the switch factor for IgG1 in humans25 and IgG2a in the mouse.26 To these was added transforming growth factor-β as the switch factor for IgA,27 IL-10 a switch factor for human IgG428 and IL-21 a switch factor for human IgG1 and IgG3.29 Each of these cytokines is believed to be made by a different CD4 T cell subset.
Are there Tfh subsets?
Since specific cytokines switch B cells to express different immunoglobulin isotypes and because these inhibit each other, are there Tfh subsets that correspond to different Th subsets—Th1, Th2, Th3, Th17 and so forth? Using an IL-21 reporter mouse, Luthje and colleagues30 showed that IL-21 producing Tfh cells are multifunctional and can make IL-4 with or without IL-21, IL-21 with interferon-γ and IL-21 with IL-10. In the gut, Tsuji et al. reported that Tfh cells can arise from gut Foxp3+ cells.31 There are still uncertainties about the relationship between the development of Tfh and Th cells (Figure 2).
When and where does class switching occur?
In response to repeated ovalbumin (OVA) aerosol, the highest number of OVA-specific IgE plasma cells was detected first in the lower respiratory tract (anterior and posterior mediastinal lymph nodes) and a smaller response in the upper respiratory tract (superficial cervical and internal jugular lymph nodes); indeed IgE mRNA was also detected in the lung and trachea by northern blot.32 In humans, IgG and IgA are produced locally as evidenced by the higher proportion of specific antibody to total immunoglobulin in nasal secretions.33 Likewise, evidence for local IgE production can be found in tears.34 Patients who are allergic to bee venom may remain sensitive decades after their last exposure. In patients with seasonal allergies, IgE antibodies continue to be produced between one season and the next when re-exposure to the allergen boosts their IgE and IgG response.35
How are Tfh cells involved in IgE class switching?
IgE and other immunoglobulin isotypes are switched under the influence of T cell-derived cytokines and CD40L stimulation. Tfh cells most notably produce IL-21.30,36 IL-21 inhibits switching to IgE and IL-21R-deficient mice make high levels of IgE,37,38 while intranasal administration of recombinant IL-21 reduced the IgE response to ovalbumin as well as T-cell production of Th2 cytokines.39 IL-21 inhibits germ line epsilon transcription in IL-4 stimulated B cells.40 However, in humans, IL-21 enhanced IgE production from peripheral blood mononuclear cells cultured with IL-4 and stimulated with anti-CD40.41 IL-21 augmentation of IL-4-driven IgE switching in human B cells appears to be STAT3-dependent.42
Using a novel system that included both T-cell receptor transgenic (DO11.10) and anti-influenza haemagglutinin B-cell receptor knock in mice, Erazo and colleagues43 reported that although switching to IgE occurred in germinal centres, these cells contained the sterile IgE transcript, while IgE B cells found outside germinal centres predominantly made the mature transcript. They also showed that very few IgE-positive B cells were present in the germinal centre following immunization and proposed that few B cells switch directly to IgE in germinal centres, rather that IgG1 secreting cells that underwent somatic hypermutation before becoming high-affinity IgE B cells once outside lymph nodes. More recently they have provided evidence that direct switching via IgE results in low-affinity IgE, while high-affinity IgE required sequential switching from IgG1 positive B cells.44 This is supported by Wessemann et al.45 who proposed that most mature B cell switching to IgE is via IgG1 and that direct IgM to IgE switching is a feature of immature B cells that may bypass hypermutation and selection in germinal centres. Recently, biocore has been used to monitor IgE affinity in Cynomolgus monkeys46 and demonstrate IgE antibody affinity maturation. Additionally, the effect of IL-21 may involve the transcription factor, Bcl6, which rescues B cells from IL-21 induced cell death47 and is expressed at low levels in plasma cells compared with germinal centre B cells, suggesting that switching to IgE may obey different rules at different stages of B-cell development.48
An alternative view has been proposed by Talay and colleagues49 who used green fluorescent protein linked to membrane form of IgE and found that most of the IgE detected in their model following Nippostrongylus braziliensis infection or immunization with TNP-OVA was directly from IgE positive germinal centre B cells. Subsequently, Yang et al.50 have used another model in which the membrane form of IgE is linked to ‘Venus’, a yellow fluorescent protein with increased sensitivity.51 These authors also found that B cells that expressed IgE differentiated into both germinal centre B cells and IgE-positive plasma cells. IgE-switched B cells with the characteristics of, and located in, the germinal centre cells were identified. IgE memory B cells rather than IgG1 memory B cells appeared to be the major contributor to B-cell IgE memory. Mice, in which the gamma 1 switch is mutated, showed that switching to IgE could still occur and that serum Nippostrongylus-specific IgE levels were unaffected.52 Furthermore, removal of Sγ1 increased the amount of OVA-specific IgE made by immunized mice.53 In vitro, both sequential switching to IgE54,55 and direct IgM to IgE switching have been reported.56 Thus, it is still unclear which mechanism is dominant and under what circumstances. A confounding factor may be B- and T-cell attenuator57 that is highly expressed on CXCR5+ Tfh cells and inhibits their capacity to produce IL-21.58
How do humans compare with mice?
Investigation into the development of allergy in humans is limited by access to the relevant tissues and almost exclusively relies on measurements of peripheral blood cells or soluble factors. Patients with atopic dermatitis do have circulating IgE secreting plasma cells.59 Furthermore, we know that making an IgE response to foods such as egg is common in industrialized countries60 and that these IgE antibodies give rise to symptoms. Like our laboratory mice, ovalbumin is not a typical allergen and continued exposure induces tolerance. Although IgE responses to egg take longer to decline in infants than in mice, the majority have little IgE antibody to egg by the time they are 2–3 years old and clinically most infants outgrow their egg allergy. The IgE response to inhalant allergens does not really start until 4–5 years of age and requires repeated low dose exposure and plenty of opportunity for the inflammation triggered by IgE bearing mast cells and its interaction with inflammatory Th2 cells. This is very difficult to recapitulate in animal models.
Considerable progress has been made to understand the regulation of the allergic immune response. We know that the two main drivers of disease are Th2 CD4 T cells and IgE attached to mast cells and basophils. We have a good understanding of how Th2 cells arise and the factors that regulate their differentiation. What has proved more challenging is to understand how and where IgE responses take place. Similarly, there are still questions about the relationship between effector Th1 and Th2 cells and Tfh cells that have yet to be adequately resolved. The life history of IgE memory B cells and the location of IgE plasma cells in allergic patients require further study. We need to know what happens with real allergens such as grass pollen and dust mite. Nevertheless, we have an exciting range of tools at our disposal and it seems reasonable to expect that these questions will be resolved over the next decade.
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Kemeny, D. The role of the T follicular helper cells in allergic disease. Cell Mol Immunol 9, 386–389 (2012). https://doi.org/10.1038/cmi.2012.31
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