Germinal center TFH cells: T(w)o be or not t(w)o be, IL-6 is the answer

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Science Immunology  13 Sep 2019:
Vol. 4, Issue 39, eaay7668
DOI: 10.1126/sciimmunol.aay7668


IL-6 inhibits the expression of IL-2Rβ (CD122) in germinal center TFH cells to maintain IL-2 hyporesponsiveness (see the related Research Article by Papillion et al.).

Follicular helper T (TFH) cells are a subpopulation of effector CD4+ T cells specialized in regulating antibody affinity maturation in the germinal center (GC). These cells also support the generation of long-lived plasma cells and memory B cells as an output of the GC reaction (1). TFH cells are not only essential to mounting high-affinity antibodies during infection but also required to support the formation of the GC-derived humoral memory responses that underlie effective vaccination (1). However, excessive numbers of TFH cells can lead to the production of autoantibodies and promote autoimmune disease development (2). Therefore, normal TFH cell differentiation is a key factor in maintaining protective immune responses.

In this issue of Science Immunology, Papillion et al. (3) unravel a paradox of TFH biology by revealing that interleukin-6 (IL-6) limits the responsiveness of TFH cells to IL-2 by intrinsically suppressing IL-2Rβ (CD122) expression in TFH cells (Fig. 1). IL-2 has been characterized as a pivotal negative regulator that inhibits TFH generation and, consequently, GC formation in mouse models (4). Other studies have shown that TFH cells produce larger amounts of IL-2 compared with that of non–TFH effector cells (5, 6). These findings raise an important but poorly understood question in GC biology addressed by this new study (3): How do TFH cells shield themselves from responding to the inhibitory signal triggered by autocrine IL-2?

For many years, IL-6 has been recognized as a major cytokine that promotes the generation of TFH cells (1), but the underlying mechanism was not well understood. Papillion et al. aimed to dissect the role of IL-6 by comparing TFH generation in wild-type (WT) and IL-6–deficient mice during influenza virus infection. Consistent with previous reports (1, 7), IL-6 was indispensable for the maintenance of TFH cells during the peak of the primary response (day 10 to day 15). However, during early influenza infection (day 7), the frequency and numbers of influenza nucleoprotein (NP)–specific TFH cells remained unchanged in the absence of IL-6. To validate the requirement of IL-6 for maintenance rather than the initiation of TFH cell responses, the authors used influenza virus expressing ovalbumin (OVA) peptide and OVA-specific OT II CD4+ T cells to show that adoptive transfer into WT or IL-6–deficient recipient mice resulted in OT II CD4+ T cells that generated comparable numbers of TFH cells 3 days after being primed by OVA-expressing influenza. These early primed OT II CD4+ T cells were purified from WT or IL-6–deficient mice and transferred into WT or IL-6–deficient recipients that had been infected with influenza virus 5 days previously and were analyzed at day 11 after infection. OT II CD4+ T cells differentiated poorly into TFH cells in IL-6–deficient recipients compared with WT recipients, confirming that IL-6 is required specifically for maintenance of TFH cells at the peak of infection.

Does IL-6 sustain TFH cell maintenance in a T cell–intrinsic manner? By generating WT/IL-6R−/− mixed bone marrow chimeric mice, the authors showed that the lack of IL-6 signaling in T cells impaired TFH cell generation at day 10 after infection. GC-TFH cells (PD-1hi), rather than non-GC TFH cells (PD-1lo), were particularly affected by the lack of T cell–intrinsic IL-6 signaling. Collectively, these results revealed an essential role for IL-6 in the formation of GC-TFH cells.

In T helper 17 (TH17) cells, IL-6 was shown to induce the phosphorylation of signal transducer and activator of transcription 3 (STAT3), which counteracts IL-2–induced STAT5 activation and promotes TH17 differentiation (8). The IL-2–STAT5 pathway was previously reported to potently suppress TFH differentiation (1). Using this influenza model in IL-2 reporter mice, the authors observed a marked up-regulation of IL-2 production by CD4+ T cells after 1 week of infection. Within the CD4+ T cell population, TFH cells were enriched for IL-2 production, with >70% of CXCR5hiPD-1hi GC-TFH cells producing IL-2 at the peak of infection. These data suggested a model in which IL-6 protects GC-TFH cells from the inhibition induced by a large amount of autocrine IL-2.

The authors then used several approaches to examine the model that IL-6 signaling is necessary for TFH generation in a high–IL-2 environment. During the early stages of influenza infection (day 7), IL-2 production was not high, and IL-6 was not essential for the development of TFH cells, as demonstrated by the administration of antibodies to IL-6/R or IL-2 (<15,000 U), which did not affect TFH differentiation. However, the combination of a low dose of IL-2 (7500 U) and antibodies to IL-6/R resulted in a marked reduction of influenza-specific TFH cells, indicating that the absence of IL-6 signaling reduced the threshold of IL-2 required for TFH cell inhibition. The authors then increased IL-2 availability through the inducible depletion of IL-2–sequestering regulatory T cells, in which TFH cells were largely diminished and further reduced in mice treated with antibodies to IL-6/R. Furthermore, using both WT/IL-6R−/− mixed bone marrow chimeras and an in vitro culture assay, the authors demonstrated that IL-6 intrinsically safeguards Bcl-6 expression and TFH differentiation by preventing responses to IL-2.

Can IL-6 prevent responses to IL-2 beyond the previously reported mechanism in which IL-6 activates STAT3 to compete with STAT5 activated by IL-2 for same binding sites (8)? IL-2 acts on cells expressing either the high-affinity trimeric IL-2R—which consists of CD25 (IL-2Rα), CD122 (IL-2Rβ), and CD132 (the common cytokine receptor γ-chain)—or the low-affinity dimeric IL-2R, which consists of only CD122 and CD132 (9). The authors found that CD25 was undetectable in both WT and IL-6−/− influenza-specific TFH cells. By contrast, CD122 was selectively increased on IL-6−/− influenza–specific and total GC-TFH cells compared with WT counterparts. Using the WT/IL-6R−/− mixed bone marrow chimera mice, the authors demonstrated that IL-6 signaling intrinsically limited the expression of CD122. Consistent with the in vivo results, IL-6 did not suppress the CD25 expression but down-regulated CD122 expression on activated CD4+ T cells cultured in vitro with antibody to IL-2. Importantly, Bcl-6 was selectively expressed in the CD25loCD122lo population, but not the CD25loCD122hi population, of activated CD4+ T cells cultured in vitro with both IL-6 and antibody to IL-2. Collectively, this evidence revealed a previously unknown mechanism of negative regulation of CD122 through IL-6 signaling.

Does excessive CD122 expression, because of the lack of IL-6 signaling, have functional consequences? By examining STAT5 signaling, the authors found that CD122 up-regulation in the absence of IL-6 was associated with remarkably higher STAT5 phosphorylation in TFH cells from IL-6−/− mice compared with WT mice after influenza infection. Furthermore, using chimeric mice generated with mixed WT and STAT5a/bfl/fl-Cd4Cre bone marrow cells, the authors found that STAT5 deficiency led to enhanced TFH differentiation and totally abrogated TFH cell suppression by antibody to IL-6/R. These results suggest that IL-6 promotes GC-TFH cells through down-regulation of CD122, which limits STAT5 signaling.

How does IL-6 suppress CD122 expression? The authors performed transposase-accessible chromatin-sequencing (ATAC-seq) using OT II CXCR5 effector T (TEFF) cells and TFH cells purified from WT or IL-6−/− mice infected with OVA-expressing influenza virus. Chromatin accessibility at the IL2rb locus of the TFH cells from IL-6−/− mice appeared to be distinct from that of the TFH cells from WT mice but similar to that of the TEFF cells from WT mice. To investigate the transcriptional regulation of the IL2rb gene, the authors mined the published data for chromatin immunoprecipitation sequencing (ChIP-seq) in CD4+ T cells (8) and identified highly overlapping STAT5 and STAT3 binding sites at the IL2rb locus. Whereas IL-6 treatment of in vitro–cultured cells substantially reduced STAT5 binding, it promoted the association of STAT3 to the IL2rb locus, which was consistent with the reported DNA binding competition between STAT3 and STAT5 for target genes. Together, data from these molecular assays suggest a model in which IL-6–induced STAT3 activation competes with IL-2–induced STAT5 to prevent transcriptional up-regulation of CD122 in TFH cells.

This study by Papillion et al. uncovered a previously unappreciated role of IL-6 in sustaining GC-TFH cells during the peak and maintenance of influenza infection. This supportive function of IL-6 is likely mediated by the induction of hyporesponsiveness to IL-2 in TFH cells after the priming phase. Data in this study also provided molecular insights into the suppression of IL2rb gene transcription by IL-6 signaling, which likely occurs, at least partially, through the prevention of STAT5 binding to the IL2rb locus. Future studies are needed to investigate whether IL-6 is superior to other STAT3-inducing cytokines or whether individual cytokines may play dominant or supplementary roles in a model-dependent manner. In addition to the frequency of GC-TFH cells focused by this study, it is also of interest to examine how IL-6 regulates the function of GC-TFH cells.

Fig. 1 GC-TFH cells highly produce IL-2 but maintain IL-2 hyporesponsiveness.

Upon primed by antigens in T cell zone, naïve CD4+ T cells can differentiate into CXCR5 effector T (TEFF) cells or CXCR5+ follicular helper T (TFH) cells that migrate into B cell follicles. TFH cells further differentiate and locate in the GC to regulate antibody affinity maturation and humoral memory formation. Sustained antigen presentation to TFH and GC-TFH cells leads to abundant production of IL-2, which potentially inhibits TFH generation through this autocrine loop. In the mouse model of influenza infection, IL-6 signaling in TFH cells activates STAT3, which competes with IL-2–induced STAT5 for binding to the Il2rβ locus. By inhibiting CD122 (IL-2Rβ) expression on TFH cells, IL-6 reduces the cellular responsiveness to IL-2 and promotes the generation of GC-TFH cells.

Credit. A. Kitterman/Science Immunology


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