Differential requirements for IRF4 in the clonal expansion and homeostatic proliferation of naive and memory murine CD8+ T cells.

Interferon regulatory factor 4 (IRF4) has critical roles in immune cell differentiation and function and is indispensable for clonal expansion and effector function in T cells. Here, we demonstrate that the AKT pathway is impaired in murine CD8+ T cells lacking IRF4. The expression of phosphatase and tensin homolog (PTEN), a negative regulator of the AKT pathway, was elevated in Irf4-/- CD8+ T cells. Inhibition of PTEN partially rescued downstream events, suggesting that PTEN constitutes a checkpoint in the IRF4-mediated regulation of cell signaling. Despite the clonal expansion defect, in the absence of IRF4, memory-like CD8+ T cells could be generated and maintained, although unable to expand in recall responses. The homeostatic proliferation of naïve Irf4-/- CD8+ T cells was impaired, whereas their number eventually reached a level similar to that of wild-type CD8+ T cells. Conversely, memory-like Irf4-/- CD8+ T cells underwent homeostatic proliferation in a manner similar to that of wild-type memory CD8+ T cells. These results suggest that IRF4 regulates the clonal expansion of CD8+ T cells at least in part via the AKT signaling pathway. Moreover, IRF4 regulates the homeostatic proliferation of naïve CD8+ T cells, whereas the maintenance of memory CD8+ T cells is IRF4-independent.

Interleukin-27-Producing CD4(+) T Cells Regulate Protective Immunity during Malaria Parasite Infection.

Interleukin-27 (IL-27) is a heterodimeric regulatory cytokine of the IL-12 family, which is produced by macrophages, dendritic cells, and B cells upon stimulation through innate immune receptors. Here, we described regulatory CD4(+) T cells that produce IL-27 in response to T cell receptor stimulation during malaria infection, inhibiting IL-2 production and clonal expansion of other T cells in an IL-27-dependent manner. IL-27-producing CD4(+) T cells were Foxp3(-)CD11a(+)CD49d(+) malaria antigen-specific CD4(+) T cells and were distinct from interferon-γ (IFN-γ) producing Th1 or IL-10 producing Tr1 cells. In mice lacking IL-27 in T cells, IL-2 production was restored and clonal expansion and IFN-γ production by specific CD4(+) T cells were improved, culminating in reduced parasite burden. This study highlights a unique population of IL-27 producing regulatory CD4(+) T cells and their critical role in the regulation of the protective immune response against malaria parasites.

IRF4 in dendritic cells inhibits IL-12 production and controls Th1 immune responses against Leishmania major.

IRF4 is a transcription factor from the IRF factor family that plays pivotal roles in the differentiation and function of T and B lymphocytes. Although IRF4 is also expressed in dendritic cells (DCs) and macrophages, its roles in these cells in vivo are not clearly understood. In this study, conditional knockout mice that lack IRF4 in DCs or macrophages were generated and infected with Leishmania major. Mice lacking DC expression of IRF4 showed reduced footpad swelling compared with C57BL/6 mice, whereas those lacking IRF4 in macrophages did not. Mice with IRF4-deficient DCs also showed reduced parasite burden, and their CD4(+) T cells produced higher levels of IFN-γ in response to L. major Ag. In the draining lymph nodes, the proportion of activated CD4(+) T cells in these mice was similar to that in the control, but the proportion of IFN-γ-producing cells was increased, suggesting a Th1 bias in the immune response. Moreover, the numbers of migrating Langerhans cells and other migratory DCs in the draining lymph nodes were reduced both before and postinfection in mice with IRF4 defects in DCs, but higher levels of IL-12 were observed in IRF4-deficient DCs. These results imply that IRF4 expression in DCs inhibits their ability to produce IL-12 while promoting their migratory behavior, thus regulating CD4(+) T cell responses against local infection with L. major.

Smad2/3 and IRF4 play a cooperative role in IL-9-producing T cell induction.

IL-9 is a pleiotropic cytokine that can regulate autoimmune and allergic responses. Th9 cells can develop from naive T cells or Th2 cells through stimulation by TGF-ß in vitro. In this study, we demonstrated that Smad2 and Smad3 are necessary for IL-9 production from T cells in an OVA-induced asthma model using T cell-specific Smad2- and Smad3-deficient mice. Smad2 and Smad3 were also redundantly essential for TGF-ß signaling to induce histone modifications for Il9 transcription. Although Smad2/3 was recruited to the Il9 promoter by TGF-ß stimulation, they are not sufficient to activate the Il9 promoter. By the screening the transcription factors, we found that IFN regulatory factor 4 (IRF4) was essential for the Smad2/3-mediated Il9 promoter activation. In addition, Smad2/3 physically interacted with IRF4, and Smad2/3 did not bind to the Il9 promoter and could not induce Th9 in IRF4-deficient T cells. Similarly, IRF4 could not stimulate Il9 transcription in the absence of Smad2/3, and TGF-ß enhanced IRF4 recruitment to the Il9 promoter in a Smad2/3-dependent manner. We propose that Smad2/3 and IRF4 cooperatively transactivate the Il9 promoter and play an important role in regulating allergic immune responses by inducing Th9 cells.

CD8+ T cells specific for a malaria cytoplasmic antigen form clusters around infected hepatocytes and are protective at the liver stage of infection.

Following Anopheles mosquito-mediated introduction into a human host, Plasmodium parasites infect hepatocytes and undergo intensive replication. Accumulating evidence indicates that CD8(+) T cells induced by immunization with attenuated Plasmodium sporozoites can confer sterile immunity at the liver stage of infection; however, the mechanisms underlying this protection are not clearly understood. To address this, we generated recombinant Plasmodium berghei ANKA expressing a fusion protein of an ovalbumin epitope and green fluorescent protein in the cytoplasm of the parasite. We have shown that the ovalbumin epitope is presented by infected liver cells in a manner dependent on a transporter associated with antigen processing and becomes a target of specific CD8(+) T cells from the T cell receptor transgenic mouse line OT-I, leading to protection at the liver stage of Plasmodium infection. We visualized the interaction between OT-I cells and infected hepatocytes by intravital imaging using two-photon microscopy. OT-I cells formed clusters around infected hepatocytes, leading to the elimination of the intrahepatic parasites and subsequent formation of large clusters of OT-I cells in the liver. Gamma interferon expressed in CD8(+) T cells was dispensable for this protective response. Additionally, we found that polyclonal ovalbumin-specific memory CD8(+) T cells induced by de novo immunization were able to confer sterile protection, although the threshold frequency of the protection was relatively high. These studies revealed a novel mechanism of specific CD8(+) T cell-mediated protective immunity and demonstrated that proteins expressed in the cytoplasm of Plasmodium parasites can become targets of specific CD8(+) T cells during liver-stage infection.

CD19(+) B cells confer protection against experimental cerebral malaria in semi-immune rodent model.

In African endemic area, adults are less vulnerable to cerebral malaria than children probably because of acquired partial immunity or semi-immune status. Here, we developed an experimental cerebral malaria (ECM) model for semi-immune mice. C57BL/6 (B6) mice underwent one, two and three cycles of infection and radical treatment (1-cure, 2-cure and 3-cure, respectively) before being finally challenged with 10(4) Plasmodium berghei ANKA without treatment. Our results showed that 100% of naïve (0-cure), 67% of 1-cure, 37% of 2-cure and none of 3-cure mice succumbed to ECM within 10 days post challenge infection. In the protected 3-cure mice, significantly higher levels of plasma IL-10 and lower levels of IFN-γ than the others on day 7 post challenge infection were observed. Major increased lymphocyte subset of IL-10 positive cells in 3-cure mice was CD5(-)CD19(+) B cells. Passive transfer of splenic CD19(+) cells from 3-cure mice protected naïve mice from ECM. Additionally, aged 3-cure mice were also protected from ECM 12 and 20 months after the last challenge infection. In conclusion, mice became completely resistant to ECM after three exposures to malaria. CD19(+) B cells are determinants in protective mechanism of semi-immune mice against ECM possibly via modulatory IL-10 for pathogenic IFN-γ production.

Accumulation of major histocompatibility complex class II(+)CD11c(-) non-lymphoid cells in the spleen during infection with Plasmodium yoelii is lymphocyte-dependent.

The spleen is the main organ for immune defense during infection with Plasmodium parasites and splenomegaly is one of the major symptoms of such infections. Using a rodent model of Plasmodium yoelii infection, MHC class II(+)CD11c(-) non-T, non-B cells in the spleen were characterized. Although the proportion of conventional dendritic cells was reduced, that of MHC II(+)CD11c(-) non-T, non-B cells increased during the course of infection. The increase in this subpopulation was dependent on the presence of lymphocytes. Experiments using Rag-2(-/-) mice with adoptively transferred normal spleen cells indicated that these cells were non-lymphoid cells; however, their accumulation in the spleen during infection with P. yoelii depended on lymphocytes. Functionally, these MHC II(+)CD11c(-) non-T, non-B cells were able to produce the proinflammatory cytokines alpha tumor necrosis factor and interleukin-6 in response to infected red blood cells, but had only a limited ability to activate antigen-specific CD4(+) T cells. This study revealed a novel interaction between MHC II(+)CD11c(-) non-lymphoid cells and lymphoid cells in the accumulations of these non-lymphoid cells in the spleen during infection with P. yoelii.

Development of memory CD8+ T cells and their recall responses during blood-stage infection with Plasmodium berghei ANKA.

Conditions required for establishing protective immune memory vary depending on the infecting microbe. Although the memory immune response against malaria infection is generally thought to be relatively slow to develop and can be lost rapidly, experimental evidence is insufficient. In this report, we investigated the generation, maintenance, and recall responses of Ag-specific memory CD8(+) T cells using Plasmodium berghei ANKA expressing OVA (PbA-OVA) as a model system. Mice were transferred with OVA-specific CD8(+) T (OT-I) cells and infected with PbA-OVA or control Listeria monocytogenes expressing OVA (LM-OVA). Central memory type OT-I cells were maintained for >2 mo postinfection and recovery from PbA-OVA. Memory OT-I cells produced IFN-γ as well as TNF-α upon activation and were protective against challenge with a tumor expressing OVA, indicating that functional memory CD8(+) T cells can be generated and maintained postinfection with P. berghei ANKA. Cotransfer of memory OT-I cells with naive OT-I cells to mice followed by infection with PbA-OVA or LM-OVA revealed that clonal expansion of memory OT-I cells was limited during PbA-OVA infection compared with expansion of naive OT-I cells, whereas it was more rapid during LM-OVA infection. The expression of inhibitory receptors programmed cell death-1 and LAG-3 was higher in memory-derived OT-I cells than naive-derived OT-I cells during infection with PbA-OVA. These results suggest that memory CD8(+) T cells can be established postinfection with P. berghei ANKA, but their recall responses during reinfection are more profoundly inhibited than responses of naive CD8(+) T cells.

Interferon regulatory factor-4 activates IL-2 and IL-4 promoters in cooperation with c-Rel.

Interferon regulatory factor (IRF)-4 is a member of the IRF transcription factor family, whose expression is primarily restricted to lymphoid and myeloid cells. In T-cells, IRF-4 expression is induced by T-cell receptor (TCR) cross-linking or treatment with phorbol-12-myristate-13-acetate (PMA)/Ionomycin, and IRF-4 is thought to be a critical factor for various functions of T-cells. To elucidate the IRF-4 functions in human adult T-cell leukemia virus type 1 (HTLV-1)-infected T-cells, which constitutively express IRF-4, we isolated IRF-4-binding proteins from T-cells, using a tandem affinity purification (TAP)-mass spectrometry strategy. Fourteen proteins were identified in the IRF-4-binding complex, including endogenous IRF-4 and the nuclear factor-kappaB (NF-κB) family member, c-Rel. The specific association of IRF-4 with c-Rel was confirmed by immunoprecipitation experiments, and IRF-4 was shown to enhance the c-Rel-dependent binding and activation of the interleukin-4 (IL-4) promoter region. We also demonstrated that IL-2 production was also enhanced by exogenously-expressed IRF-4 and c-Rel in the presence of P/I, in T-cells, and that the optimal IL-2 and IL-4 productions in vivo was IRF-4-dependent using IRF-4-/- mice. These data provide molecular evidence to support the clinical observation that elevated expression of c-Rel and IRF-4 is associated with the prognosis in adult T-cell leukemia/lymphoma (ATLL) patients, and present possible targets for future gene therapy.

Production of IFN-γ by CD4(+) T cells in response to malaria antigens is IL-2 dependent.

T-cell immune responses are critical for protection of the host and for disease pathogenesis during infection with Plasmodium species. We examined the regulation of CD4(+) T-cell cytokine responses during infection with Plasmodium berghei ANKA (PbA). CD4(+) T cells from PbA-infected mice produced IFN-γ, IL-4 and IL-10 in response to TCR stimulation at levels higher than those from uninfected mice. This altered cytokine response was dependent on parasitemia. To examine the specificity of the response, mice were adoptively transferred with CD4(+) T cells from OT-II TCR transgenic mice and were infected with PbA expressing OVA. Unexpectedly, CD4(+) T cells from the OT-II-transferred wild-type PbA-infected mice showed high levels of IFN-γ production after stimulation with OVA and the cells producing IFN-γ were not OT-II but were host CD4(+) T cells. Further investigation revealed that host CD4(+) T cells produced IFN-γ in response to IL-2 produced by activated OT-II cells. This IFN-γ response was completely inhibited by anti-CD25 mAbs, and this effect was not due to the block of the survival signals provided by IL-2. Furthermore, IFN-γ production by CD4(+) T cells in response to PbA antigens was dependent on IL-2. These findings suggest the importance of IL-2 levels during infection with malaria parasites and indicate that CD4(+) T cells can produce IFN-γ without TCR engagement via a bystander mechanism in response to IL-2 produced by other activated CD4(+) T cells.

The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection.

Polarization of macrophages to M1 or M2 cells is important for mounting responses against bacterial and helminth infections, respectively. Jumonji domain containing-3 (Jmjd3), a histone 3 Lys27 (H3K27) demethylase, has been implicated in the activation of macrophages. Here we show that Jmjd3 is essential for M2 macrophage polarization in response to helminth infection and chitin, though Jmjd3 is dispensable for M1 responses. Furthermore, Jmjd3 (also known as Kdm6b) is essential for proper bone marrow macrophage differentiation, and this function depends on demethylase activity of Jmjd3. Jmjd3 deficiency affected trimethylation of H3K27 in only a limited number of genes. Among them, we identified Irf4 as encoding a key transcription factor that controls M2 macrophage polarization. Collectively, these results show that Jmjd3-mediated H3K27 demethylation is crucial for regulating M2 macrophage development leading to anti-helminth host responses.

Interferon regulatory factor 4 differentially regulates the production of Th2 cytokines in naive vs. effector/memory CD4+ T cells.

Interferon regulatory factor (IRF) 4 is a member of the IRF family of transcription factors and plays critical roles in the development of CD4(+) T cells into Th2 and Th17 cells. Using the infection model of Nippostrongyrus brasiliensis, we have confirmed the critical roles of IRF-4 in Th2 development in vivo by using IRF-4(-/-) BALB/c mice. However, naïve IRF-4(-/-)CD4(+) T cells produced Th2 cytokines, including IL-4, IL-5, and IL-10, but not IL-2 or IFN-gamma, at levels higher than wild-type BALB/c CD4(+) T cells in response to T cell receptor stimulation. In contrast, effector/memory IRF-4(-/-)CD4(+) T cells did not exhibit increased production of Th2 cytokines. Knockdown of IRF-4 expression by using small interfering RNA promoted IL-4 production in naïve CD4(+) T cells but inhibited it in effector/memory CD4(+) T cells. These results indicate that IRF-4 plays differential roles in the regulation of Th2 cytokine production in naïve CD4(+) T cells and effector/memory CD4(+) T cells. IRF-4 inhibits Th2 cytokine production in naïve CD4(+) T cells, whereas it promotes Th2 cytokine production in effector/memory CD4(+) T cells.

Malaria-specific and nonspecific activation of CD8+ T cells during blood stage of Plasmodium berghei infection.

Cerebral malaria is one of the severe complications of Plasmodium falciparum infection. Studies using a rodent model of Plasmodium berghei ANKA infection established that CD8(+) T cells are involved in the pathogenesis of cerebral malaria. However, it is unclear whether and how Plasmodium-specific CD8(+) T cells can be activated during the erythrocyte stage of malaria infection. We generated recombinant Plasmodium berghei ANKA expressing OVA (OVA-PbA) to investigate the parasite-specific T cell responses during malaria infection. Using this model system, we demonstrate two types of CD8(+) T cell activations during the infection with malaria parasite. Ag (OVA)-specific CD8(+) T cells were activated by TAP-dependent cross-presentation during infection with OVA-PbA leading to their expression of an activation phenotype and granzyme B and the development to functional CTL. These highly activated CD8(+) T cells were preferentially sequestered in the brain, although it was unclear whether these cells were involved in the pathogenesis of cerebral malaria. Activation of OVA-specific CD8(+) T cells in RAG2 knockout TCR-transgenic mice during infection with OVA-PbA did not have a protective role but rather was pathogenic to the host as shown by their higher parasitemia and earlier death when compared with RAG2 knockout mice. The OVA-specific CD8(+) T cells, however, were also activated during infection with wild-type parasites in an Ag-nonspecific manner, although the levels of activation were much lower. This nonspecific activation occurred in a TAP-independent manner, appeared to require NK cells, and was not by itself pathogenic to the host.

Interferon regulatory factor 4 negatively regulates the production of proinflammatory cytokines by macrophages in response to LPS.

A member of the IFN regulatory factor (IRF) family of transcription factors, IRF-4 is expressed in lymphocytes and macrophage/dendritic cells. Studies using IRF-4-deficient mice have revealed the critical roles of IRF-4 in lymphocyte responses. However, the role of IRF-4 in innate immune responses is not clearly understood. Here, we demonstrate that IRF-4 negatively regulates the production of proinflammatory cytokines by macrophages in response to Toll-like receptor (TLR) stimulation. Mice lacking IRF-4 are sensitive to LPS-induced shock, and their macrophages produce high levels of proinflammatory cytokines, including TNF-alpha and IL-6, in response to TLR ligands. The inhibitory role of IRF-4 in response to TLR stimulation was confirmed by the down-regulation of IRF-4 expression in normal macrophages by using the small interfering RNA technique and by the overexpression of IRF-4 in macrophage line RAW264.7. Activation of the important signaling pathways for cytokine production, NF-kappaB and JNK (c-Jun N-terminal kinase), was enhanced after LPS stimulation in IRF-4(-/-) macrophages. These results imply that IRF-4 negatively regulates TLR signaling and is inhibitory to the production of proinflammatory cytokines in response to TLR stimulation.

Negative regulation of Toll-like-receptor signaling by IRF-4.

The recognition of microbial components by Toll-like receptors (TLRs) is an event central to the activation of innate and adaptive immune systems. TLR activation triggers the induction of downstream target genes, wherein the TLR-interacting adaptor molecule MyD88 recruits various signaling molecules and transcription factors. Two members of the IFN regulatory factor (IRF) family of transcription factors, IRF-5 and IRF-7, interact with MyD88 and induce proinflammatory cytokines and type I IFNs, respectively. Here, we show that IRF-4 also interacts with MyD88 and acts as a negative regulator of TLR signaling. IRF-4 mRNA is induced by TLR activation, and IRF-4 competes with IRF-5, but not with IRF-7, for MyD88 interaction. The TLR-dependent induction of proinflammatory cytokines is markedly enhanced in peritoneal macrophages from mice deficient in the Irf4 gene, whereas the induction is inhibited by the ectopic expression of IRF-4 in a macrophage cell line. The critical function of IRF-4 in TLR signaling in vivo is underscored by the observation that Irf4-deficient mice show hypersensitivity to DNA-induced shock, with elevated serum proinflammatory cytokine levels. This study may provide an insight into the complex regulatory mechanisms of MyD88 signaling by IRFs.

Active repression of IFN regulatory factor-1-mediated transactivation by IFN regulatory factor-4.

IFN regulatory factor-4 (IRF-4) is a transcription factor that is involved in the development and the functions of lymphocytes, macrophages and dendritic cells. Despite their critical roles in immune system regulation, the target genes controlled by IRF-4 are poorly understood. In this study, we determined the consensus DNA-binding sequences preferred for IRF-4 by in vitro binding site selections. IRF-4 preferentially bound to the sequences containing tandem repeats of 5'-GAAA-3', flanked by CpC, in most cases. IRF-4 repressed the promoter bearing tandem copies of the selected binding sequence, while IRF-1 activated the same constructs. Interestingly, the IRF-1-dependent transactivation is inhibited in the presence of IRF-4, but not IRF-2. A series of deletion mutants of IRF-4 revealed that its DNA-binding domain was necessary and sufficient to antagonize the IRF-1-dependent transactivation. This dominant negative action of IRF-4 over IRF-1 was also observed in a natural promoter context, such as the TRAIL gene. These results indicate that IRF-4 acts as a natural antagonist against IRF-1 in immune cells.

Critical roles of interferon regulatory factor 4 in CD11bhighCD8alpha- dendritic cell development.

IFN regulatory factors (IRFs) are a family of transcription factors that play an essential role in the homeostasis and function of immune systems. Recent studies indicated that IRF-8 is critical for the development of CD11b(low)CD8alpha(+) conventional dendritic cells (DCs) and plasmacytoid DCs. Here we show that IRF-4 is important for CD11b(high)CD8alpha(-) conventional DCs. The development of CD11b(high) DCs from bone marrow of IRF-4(-/-) mice was severely impaired in two culture systems supplemented with either GM-CSF or Flt3-ligand. In the IRF-4(-/-) spleen, the number of CD4(+)CD8alpha(-) DCs, a major subset of CD11b(high) DCs, was severely reduced. IRF-4 and IRF-8 were expressed in the majority of CD11b(high)CD4(+)CD8alpha(-) DCs and CD11b(low)CD8alpha(+) DCs, respectively, in a mutually exclusive manner. These results imply that IRF-4 and IRF-8 selectively play critical roles in the development of the DC subsets that express them.

Merozoite surface protein 1-specific immune response is protective against exoerythrocytic forms of Plasmodium yoelii.

One of the difficulties in developing an effective malaria vaccine is the antigenic change of the parasite during the life cycle. It is desirable that vaccine-induced protective immunity be effective at different stages of parasite development. Merozoite surface protein 1 (MSP1) is a candidate vaccine antigen against blood-stage malaria, but it is also expressed in the exoerythrocytic forms. It was not known, however, whether the anti-MSP1 immune response is effective against the liver-stage malaria parasite. We generated a recombinant protein of MSP1 fused to heat-shock cognate protein 70 (hsc70) and studied its vaccination effect. When C57BL/6 mice were immunized with the fusion protein prior to challenge infection with Plasmodium yoelii sporozoites, the onset of parasitemia was delayed or no parasitemia was observed. To determine whether this was due to the protective immunity against liver-stage parasites, P. yoelii-specific rRNA in the infected liver was quantitated by real-time reverse transcription-PCR analysis. The level of parasite-specific rRNA was reduced in mice immunized with the fusion protein of MSP1 and hsc70 but not with hsc70 alone, indicating that MSP1-specific immunity can be protective against the exoerythrocytic form of the parasite. Furthermore, the adoptive transfer experiments of immune lymphocytes and serum into naive mice suggested that the protective immunity was dependent on cellular and not humoral immunity. Finally, the vaccine-induced protection was also observed in A/J, C3H, and BALB/c mice, suggesting that MSP1-specific protective immunity at the exoerythrocytic stage can be induced in animals over a wide range of genetic backgrounds.