Magdalena Huber

The development of inflammatory T(H)-17 cells requires interferon-regulatory factor 4

Interferon-regulatory factor 4 (IRF4) is essential for the development of T helper type 2 cells. Here we show that IRF4 is also critical for the generation of interleukin 17–producing T helper cells (TH-17 cells), which are associated with experimental autoimmune encephalomyelitis. IRF4-deficient (Irf4−/−) mice did not develop experimental autoimmune encephalomyelitis, and T helper cells from such mice failed to differentiate into TH-17 cells. Transfer of wild-type T helper cells into Irf4−/− mice rendered the mice susceptible to experimental autoimmune encephalomyelitis. Irf4−/− T helper cells had less expression of RORγt and more expression of Foxp3, transcription factors important for the differentiation of TH-17 and regulatory T cells, respectively. Altered regulation of both transcription factors contributed to the phenotype of Irf4−/− T helper cells. Our data position IRF4 at the center of T helper cell development, influencing not only T helper type 2 but also TH-17 differentiation.

IRF4 is essential for IL-21-mediated induction, amplification, and stabilization of the Th17 phenotype

Differentiation of murine T-helper (Th) 17 cells is induced by antigenic stimulation and the sequential action of the cytokines IL-6, IL-21, and IL-23, along with TGFβ. Current dogma proposes that IL-6 induces IL-21, which, in a STAT3-dependent manner, amplifies its own transcription, contributes to IL-17 production, and, moreover, promotes the expression of the IL-23 receptor. This, in turn, prepares cells for IL-23-mediated stabilization of the Th17 phenotype. Here we demonstrate that these effects of IL-21 on Th17 differentiation are completely dependent on IFN regulatory factor 4 (IRF4). After culturing in the presence of IL-21 plus TGFβ, IRF4-deficient (Irf4−/−) Th cells showed a profound intrinsic defect in IL-17 production and in the autocrine IL-21 loop. Likewise, the levels of IL-23 receptor and the lineage-specific orphan nuclear receptors RORα and RORγt were diminished, whereas the T regulatory (Treg) transcription factor forkhead box P3 (Foxp3) was strongly up-regulated, consistent with the reciprocal relationship between Th17 and Treg development. Despite this loss of IL-21 functions, IL-21-induced STAT3 activation was unimpaired and induced normal Socs3 expression. Forced expression of Foxp3 in WT cells inhibited IL-21-mediated IL-17 production, suggesting that the increase in Foxp3 contributes to the Irf4−/− phenotype. Additionally, the low levels of RORα and RORγt are also partially responsible, because simultaneous overexpression of both proteins restored IL-17 production in Irf4−/− cells to some extent. These data highlight IRF4 as a decisive factor during the IL-21-mediated steps of Th17 development by influencing the balance of Foxp3, RORα, and RORγt.

A Th17‐like developmental process leads to CD8+ Tc17 cells with reduced cytotoxic activity

Activation of naive CD8+ T cells with antigen in the absence of skewing cytokines triggers their differentiation into effector CTL, which induces death of target cells. We show that CD8+ T cells activated in the presence of the cytokines IL‐6 or IL‐21 plus TGF‐β similar to CD4+ T cells, develop into IL‐17‐producing (Tc17) cells. These cells display greatly suppressed cytotoxic function along with low levels of the CTL markers: T‐box transcription factor Eomesodermin, granzyme B and IFN‐γ. Instead, these cells express hallmark molecules of Th17 program including retinoic acid receptor‐related orphan receptor (ROR)γt, RORα, IL‐21 and IL‐23R. The expression of the type 17 master regulator RORγt is causally linked to Tc17 generation, because its overexpression stimulates production of IL‐17 in the presence of IL‐6 or IL‐21. Both, upregulation of the type 17 program as well as suppression of CTL differentiation are STAT3 dependent. Furthermore, Tc17 cells producing IL‐17 but not granzyme B are also detectable in EAE, a mouse model for multiple sclerosis. Our data point to the existence of mutually exclusive CTL and Tc17 developmental pathways in vitro and in vivo.

IL-17A secretion by CD8+ T cells supports Th17-mediated autoimmune encephalomyelitis

IL-17-producing CD8+ T (Tc17) cells are detectible in multiple sclerosis (MS) lesions; however, their contribution to the disease is unknown. To identify functions of Tc17 cells, we induced EAE, a murine model of MS, in mice lacking IFN regulatory factor 4 (IRF4). IRF4-deficient mice failed to generate Tc17 and Th17 cells and were resistant to EAE. After adoptive transfer of WT CD8+ T cells and subsequent immunization for EAE induction in these mice, the CD8+ T cells developed a Tc17 phenotype in the periphery but could not infiltrate the CNS. Similarly, transfer of small numbers of WT CD4+ T cells alone did not evoke EAE, but when transferred together with CD8+ T cells, IL-17-producing CD4+ (Th17) T cells accumulated in the CNS and mice developed severe disease. Th17 accumulation and development of EAE required IL-17A production by CD8+ T cells, suggesting that Tc17 cells are required to promote CD4+ T cell-mediated induction of EAE. Accordingly, patients with early-stage MS harbored a greater number of Tc17 cells in the cerebrospinal fluid than in peripheral blood. Our results reveal that Tc17 cells contribute to the initiation of CNS autoimmunity in mice and humans by supporting Th17 cell pathogenicity.

Transcription factor IRF4 determines germinal center formation through follicular T-helper cell differentiation

Follicular T-helper (TFH) cells cooperate with GL7+CD95+ germinal center (GC) B cells to induce antibody maturation. Herein, we identify the transcription factor IRF4 as a T-cell intrinsic precondition for TFH cell differentiation and GC formation. After immunization with protein or infection with the protozoon Leishmania major, draining lymph nodes (LNs) of IFN-regulatory factor-4 (Irf4−/−) mice lacked GCs and GC B cells despite developing normal initial hyperplasia. GCs were also absent in Peyer’s patches of naive Irf4−/− mice. Accordingly, CD4+ T cells within the LNs and Peyer’s patches failed to express the TFH key transcription factor B-cell lymphoma-6 and other TFH-related molecules. During chronic leishmaniasis, the draining Irf4−/− LNs disappeared because of massive cell death. Adoptive transfer of WT CD4+ T cells or few L. major primed WT TFH cells reconstituted GC formation, GC B-cell differentiation, and LN cell survival. In support of a T-cell intrinsic IRF4 activity, Irf4−/− TFH cell differentiation was not rescued by close neighborhood to transferred WT TFH cells. Together with its known B lineage-specific roles during plasma cell maturation and class switch, our study places IRF4 in the center of antibody production toward T-cell–dependent antigens.

c-Rel is crucial for the induction of Foxp3(+) regulatory CD4(+) T cells but not T(H)17 cells

The NF‐κB/Rel family member c‐Rel was described to be required for the development of TH1 responses. However, the role of c‐Rel in the differentiation of TH17 and regulatory CD4+Foxp3+ T cells (Treg) remains obscure. Here, we show that in the absence of c‐Rel, in vitro differentiation of pro‐inflammatory TH17 cells is normal. In contrast, generation of inducible Treg (iTreg) within c‐Rel‐deficient CD4+ T cells was severely hampered and correlated to reduced numbers of Foxp3+ T cells in vivo. Mechanistically, in vitro conversion of naive CD4+ T cells into iTreg was crucially dependent on c‐Rel‐mediated synthesis of endogenous IL‐2. The addition of exogenous IL‐2 was sufficient to rescue the development of c‐Rel‐deficient iTreg. Thus, c‐Rel is essential for the development of Foxp3+ Treg but not for TH17 cells via regulating the production of IL‐2.

IRF4 at the crossroads of effector T-cell fate decision

Interferon regulatory factor 4 (IRF4) is a transcription factor that is expressed in hematopoietic cells and plays pivotal roles in the immune response. Originally described as a lymphocyte‐specific nuclear factor, IRF4 promotes differentiation of naïve CD4+ T cells into T helper 2 (Th2), Th9, Th17, or T follicular helper (Tfh) cells and is required for the function of effector regulatory T (eTreg) cells. Moreover, IRF4 is essential for the sustained differentiation of cytotoxic effector CD8+ T cells, for CD8+ T‐cell memory formation, and for differentiation of naïve CD8+ T cells into IL‐9‐producing (Tc9) and IL‐17‐producing (Tc17) CD8+ T‐cell subsets. In this review, we focus on recent findings on the role of IRF4 during the development of CD4+ and CD8+ T‐cell subsets and the impact of IRF4 on T‐cell‐mediated immune responses in vivo.

The transcription factor Interferon Regulatory Factor 4 is required for the generation of protective effector CD8+ T cells

Robust cytotoxic CD8+ T-cell response is important for immunity to intracellular pathogens. Here, we show that the transcription factor IFN Regulatory Factor 4 (IRF4) is crucial for the protective CD8+ T-cell response to the intracellular bacterium Listeria monocytogenes. IRF4-deficient (Irf4−/−) mice could not clear L. monocytogenes infection and generated decreased numbers of L. monocytogenes-specific CD8+ T cells with impaired effector phenotype and function. Transfer of wild-type CD8+ T cells into Irf4−/− mice improved bacterial clearance, suggesting an intrinsic defect of CD8+ T cells in Irf4−/− mice. Following transfer into wild-type recipients, Irf4−/− CD8+ T cells became activated and showed initial proliferation upon L. monocytogenes infection. However, these cells could not sustain proliferation, produced reduced amounts of IFN-γ and TNF-α, and failed to acquire cytotoxic function. Forced IRF4 expression in Irf4−/− CD8+ T cells rescued the defect. During acute infection, Irf4−/− CD8+ T cells demonstrated diminished expression of B lymphocyte-induced maturation protein-1 (Blimp-1), inhibitor of DNA binding (Id)2, and T-box expressed in T cells (T-bet), transcription factors programming effector-cell generation. IRF4 was essential for expression of Blimp-1, suggesting that altered regulation of Blimp-1 contributes to the defects of Irf4−/− CD8+ T cells. Despite increased levels of B-cell lymphoma 6 (BCL-6), Eomesodermin, and Id3, Irf4−/− CD8+ T cells showed impaired memory-cell formation, indicating additional functions for IRF4 in this process. As IRF4 governs B-cell and CD4+ T-cell differentiation, the identification of its decisive role in peripheral CD8+ T-cell differentiation, suggests a common regulatory function for IRF4 in adaptive lymphocytes fate decision.

Heterogeneity in the Differentiation and Function of CD8+ T Cells

It is well established that CD8+ T cells constitute an important branch of adaptive immunity contributing to clearance of intracellular pathogens and providing long-term protection. These functions are mostly fulfilled by the best characterized subpopulation of CD8+ T cells, the cytotoxic T lymphocytes (also called Tc1 cells), owing to their ability to kill infected cells and to secrete cytokines such as interferon-γ and tumor necrosis factor-α. However, there is growing evidence for alternative CD8+ T cell fates influencing CD4+ T-cell-mediated responses in the context of allergy, autoimmunity and infections. Thus, like subpopulations of CD4+ T cells, also CD8+ T cells under particular conditions acquire the expression of interleukin (IL)-4, IL-5, IL-9, IL-13, IL-17 or suppressive activity and thereby influence immune responses. The process of CD8+ T-cell differentiation is dictated by antigen strength, co-stimulatory molecules and cytokines. These environmental cues induce transcription factors further specifying CD8+ T-cell decision into Tc1, Tc2, Tc9, Tc17 or CD8+ T regulatory fate. Here, we discuss our current understanding about functional diversity of effector CD8+ T cells and contribution of transcription factors to this process.

Tc9 cells, a new subset of CD8(+) T cells, support Th2-mediated airway inflammation.

Similar to T‐helper (Th) cells, CD8+ T cells also differentiate into distinct subpopulations. However, the existence of IL‐9‐producing CD8+ T (Tc9) cells has not been elucidated so far. We show that murine CD8+ T cells activated in the presence of IL‐4 plus TGF‐β develop into transient IL‐9 producers characterized by specific IFN‐γ and IL‐10 expression patterns as well as by low cytotoxic function along with diminished expression of the CTL‐associated transcription factors T‐bet and Eomesodermin. Similarly to the CD4+ counterpart, Tc9 cells required for their differentiation STAT6 and IRF4. Tc9 cells deficient for these master regulators displayed increased levels of Foxp3 that in turn suppressed IL‐9 production. In an allergic airway disease model, Tc9 cells promoted the onset of airway inflammation, mediated by subpathogenic numbers of Th2 cells. This support was specific for Tc9 cells because CTLs failed to exert this function. We detected increased Tc9 frequency in the periphery in mice and humans with atopic dermatitis, a Th2‐associated skin disease that often precedes asthma. Thus, our data point to the existence of Tc9 cells and to their supportive function in Th2‐dependent airway inflammation, suggesting that these cells might be a therapeutic target in allergic disorders.