Hartmann Raifer

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.

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.

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.

Unlike αβ T cells, γδ T cells, LTi cells and NKT cells do not require IRF4 for the production of IL‐17A and IL‐22

Apart from conventional CD4+ Th17 cells, the cytokines IL‐17A and IL‐22 can also be produced by γδ T cells, NK cells and lymphoid tissue inducer (LTi) cells. Th17 cells develop from precursor cells after T‐cell receptor stimulation in the presence of TGF‐β, IL‐6 and IL‐23. In contrast, a subset of γδ T cells (“γδT17”) is committed for fast IL‐17 production already in the thymus; however, γδ T cells can also produce IL‐17 after prolonged in vitro stimulation via their γδ T‐cell receptor plus IL‐23. Here, we show that γδ T‐, LTi‐ and NKT cells differ extensively from Th17 cells in their signalling requirements for the generation of IL‐17A and IL‐22. While production of these cytokines by Th17 cells totally depends on the transcription factor interferon regulatory factor 4 (IRF4), IRF4 is irrelevant in the other cell types. As for γδ T cells, this finding pertains to both thymic commitment and prolonged in vitro culture. Furthermore, IL‐17A‐producing γδ T cells accumulate in the central nervous system of IRF4 deficient (Irf4−/−) mice during experimental autoimmune encephalomyelitis. IL‐17A‐producing WT and Irf4−/− γδ T cells equally express CCR6 and lack CD27. The underlying IRF4‐independent pathway partially involves STAT3 during in vitro stimulation.

Mast cells have no impact on cutaneous leishmaniasis severity and related Th2 differentiation in resistant and susceptible mice.

The genus leishmania comprises different protozoan parasites which are causative agents of muco‐cutaneous and systemic, potentially lethal diseases. After infection with the species Leishmania major, resistant mice expand Th1 cells which stimulate macrophages for Leishmania destruction. In contrast, susceptible mice generate Th2 cells which deactivate macrophages, leading to systemic spread of the pathogens. Th‐cell differentiation is determined within the first days, and Th2 cell differentiation requires IL‐4, whereby the initial IL‐4 source is often unknown. Mast cells are potential sources of IL‐4, and hence their role in murine leishmaniasis has previously been studied in mast cell‐deficient Kit mutant mice, although these mice display immunological phenotypes beyond mast cell deficiency. We therefore readdressed this question by infecting Kit‐independent mast cell‐deficient mice that are Th1 (C57BL/6 CpaCre) or Th2 (BALB/c CpaCre) prone with L. major. Using different parasite doses and intra‐ or subcutaneous infection routes, the results demonstrate no role of mast cells on lesion size development, parasite load, immune cell phenotypes expanding in draining lymph nodes, and cytokine production during murine cutaneous leishmaniasis. Thus, other cell types such as ILCs or T cells have to be considered as primary source of Th2‐driving IL‐4.

Reciprocal regulation of the Il9 locus by counteracting activities of transcription factors IRF1 and IRF4

The T helper 9 (Th9) cell transcriptional network is formed by an equilibrium of signals induced by cytokines and antigen presentation. Here we show that, within this network, two interferon regulatory factors (IRF), IRF1 and IRF4, display opposing effects on Th9 differentiation. IRF4 dose-dependently promotes, whereas IRF1 inhibits, IL-9 production. Likewise, IRF1 inhibits IL-9 production by human Th9 cells. IRF1 counteracts IRF4-driven Il9 promoter activity, and IRF1 and IRF4 have opposing function on activating histone modifications, thus modulating RNA polymerase II recruitment. IRF1 occupancy correlates with decreased IRF4 abundance, suggesting an IRF1-IRF4-binding competition at the Il9 locus. Furthermore, IRF1 shapes Th9 cells with an interferon/Th1 gene signature. Consistently, IRF1 restricts the IL-9-dependent pathogenicity of Th9 cells in a mouse model of allergic asthma. Thus our study reveals that the molecular ratio between IRF4 and IRF1 balances Th9 fate, thus providing new possibilities for manipulation of Th9 differentiation.

Antigen receptor-mediated depletion of FOXP3 in induced regulatory T-lymphocytes via PTPN2 and FOXO1.

Regulatory T-cells induced via IL-2 and TGFβ in vitro (iTreg) suppress immune cells and are potential therapeutics during autoimmunity. However, several reports described their re-differentiation into pathogenic cells in vivo and loss of their key functional transcription factor (TF) FOXP3 after T-cell antigen receptor (TCR)-signalling in vitro. Here, we show that TCR-activation antagonizes two necessary TFs for foxp3 gene transcription, which are themselves regulated by phosphorylation. Although the tyrosine phosphatase PTPN2 is induced to restrain IL-2-mediated phosphorylation of the TF STAT5, expression of the TF FOXO1 is downregulated and miR-182, a suppressor of FOXO1 expression, is upregulated. TGFβ counteracts the FOXP3-depleting TCR-signal by reassuring FOXO1 expression and by re-licensing STAT5 phosphorylation. Overexpressed phosphorylation-independent active versions of FOXO1 and STAT5 or knockdown of PTPN2 restores FOXP3 expression despite TCR-signal and absence of TGFβ. This study suggests novel targets for stabilisation and less dangerous application of iTreg during devastating inflammation.

Plasmodium falciparum Hop (PfHop) Interacts with the Hsp70 Chaperone in a Nucleotide-Dependent Fashion and Exhibits Ligand Selectivity

Heat shock proteins (Hsps) play an important role in the development and pathogenicity of malaria parasites. One of the most prominent functions of Hsps is to facilitate the folding of other proteins. Hsps are thought to play a crucial role when malaria parasites invade their host cells and during their subsequent development in hepatocytes and red blood cells. It is thought that Hsps maintain proteostasis under the unfavourable conditions that malaria parasites encounter in the host environment. Although heat shock protein 70 (Hsp70) is capable of independent folding of some proteins, its functional cooperation with heat shock protein 90 (Hsp90) facilitates folding of some proteins such as kinases and steroid hormone receptors into their fully functional forms. The cooperation of Hsp70 and Hsp90 occurs through an adaptor protein called Hsp70-Hsp90 organising protein (Hop). We previously characterised the Hop protein from Plasmodium falciparum (PfHop). We observed that the protein co-localised with the cytosol-localised chaperones, PfHsp70-1 and PfHsp90 at the blood stages of the malaria parasite. In the current study, we demonstrated that PfHop is a stress-inducible protein. We further explored the direct interaction between PfHop and PfHsp70-1 using far Western and surface plasmon resonance (SPR) analyses. The interaction of the two proteins was further validated by co-immunoprecipitation studies. We observed that PfHop and PfHsp70-1 associate in the absence and presence of either ATP or ADP. However, ADP appears to promote the association of the two proteins better than ATP. In addition, we investigated the specific interaction between PfHop TPR subdomains and PfHsp70-1/ PfHsp90, using a split-GFP approach. This method allowed us to observe that TPR1 and TPR2B subdomains of PfHop bind preferentially to the C-terminus of PfHsp70-1 compared to PfHsp90. Conversely, the TPR2A motif preferentially interacted with the C-terminus of PfHsp90. Finally, we observed that recombinant PfHop occasionally eluted as a protein species of twice its predicted size, suggesting that it may occur as a dimer. We conducted SPR analysis which suggested that PfHop is capable of self-association in presence or absence of ATP/ADP. Overall, our findings suggest that PfHop is a stress-inducible protein that directly associates with PfHsp70-1 and PfHsp90. In addition, the protein is capable of self-association. The findings suggest that PfHop serves as a module that brings these two prominent chaperones (PfHsp70-1 and PfHsp90) into a functional complex. Since PfHsp70-1 and PfHsp90 are essential for parasite growth, findings from this study are important towards the development of possible antimalarial inhibitors targeting the cooperation of these two chaperones.

β5i subunit deficiency of the immunoproteasome leads to reduced Th2 response in OVA induced acute asthma.

The immunoproteasome subunit β5i has been shown to play an important role in Th1/Th17 driven models of colitis and arthritis. However, the function of β5i in Th2 dependent diseases remains enigmatic. To study the role of β5i in Th2-driven pathology, β5i knockout (KO) and control mice were tested in different models of experimental allergic asthma. β5i-deficient mice showed reduced OVA/Alum- and subcutaneous/OVA-induced acute asthma with decreased eosinophilia in the bronchoalveolar lavage (BAL), low OVA-specific IgG1 and reduced local and systemic Th2 cytokines. While Th2 cells in the lungs were reduced, Tregs and Th1 cells were not affected. Attenuated asthma in β5i KO mice could not be attributed to defects in OVA uptake or maturation of dendritic cells in the lung. Surprisingly, β5i deficient mice developed HDM asthma which was comparable to control mice. Here, we present novel evidence for the requirement of the β5i immunosubunit to generate a strong Th2 response during OVA- but not HDM-induced acute asthma. The unexpected role of β5i in OVA asthma remains to be clarified.

Novel GM-CSF signals via IFN-γR/IRF-1 and AKT/mTOR license monocytes for suppressor function

Granulocyte-macrophage colony-stimulating factor (GM-CSF) controls proliferation and survival of myeloid cells including monocytes. Here, we describe a time-dependent licensing process driven by GM-CSF in murine Ly6Chigh and human CD14+ monocytes that disables their inflammatory functions and promotes their conversion into suppressor cells. This 2-step licensing of monocytes requires activation of the AKT/mTOR/mTORC1 signaling cascade by GM-CSF followed by signaling through the interferon-γ receptor (IFN-γR)/interferon regulatory factor-1 (IRF-1) pathway. Only licensing-dependent adaptations in Toll-like receptor/inflammasome, IFN-γR, and phosphatidylinositol 3-kinase/AKT/mTOR signaling lead to stabilized expression of inducible nitric oxide synthase by mouse and indoleamine 2,3-dioxygenase (IDO) by human monocytes, which accounts for their suppressor activity. This study suggests various myeloid cells with characteristics similar to those described for monocytic myeloid-derived suppressor cells, Mreg, or suppressor macrophages may arise from licensed monocytes. Markers of GM-CSF-driven monocyte licensing, including p-Akt, p-mTOR, and p-S6, distinguish inflammatory monocytes from potentially suppressive monocytes in peripheral blood of patients with high-grade glioma.