IL‐34‐ and M‐CSF‐induced macrophages switch memory T cells into Th17 cells via membrane IL‐1α

Macrophages orchestrate the immune response via the polarization of CD4⁺ T helper (Th) cells. Different subsets of macrophages with distinct phenotypes, and sometimes opposite functions, have been described. M‐CSF and IL‐34 induce the differentiation of monocytes into IL‐10^high IL‐12^low immunoregulatory macrophages, which are similar to tumor‐associated macrophages (TAMs) in ovarian cancer. In this study, we evaluated the capacity of human macrophages induced in the presence of M‐CSF (M‐CSF macrophages) or IL‐34 (IL‐34 macrophages) and ovarian cancer TAMs to modulate the phenotype of human CD4⁺ T cells. Taken together, our results show that M‐CSF‐, IL‐34 macrophages, and TAMs switch non‐Th17 committed memory CD4⁺ T cells into conventional CCR4⁺ CCR6⁺ CD161⁺ Th17 cells, expressing or not IFN‐gamma. Contrary, the pro‐inflammatory GM‐CSF macrophages promote Th1 cells. The polarization of memory T cells into Th17 cells is mediated via membrane IL‐1α (mIL‐1α), which is constitutively expressed by M‐CSF‐, IL‐34 macrophages, and TAMs. This study elucidates a new mechanism that allows macrophages to maintain locally restrained and smoldering inflammation, which is required in angiogenesis and metastasis.     

The inflammatory cytokine,GM‐CSF,alters the developmental outcome of murine dendritic cells

Fms‐like tyrosine kinase 3 ligand (Flt3L) is a major cytokine that drives development of dendritic cells (DCs) under steady state, whereas GM‐CSF becomes a prominent influence on differentiation during inflammation. The influence GM‐CSF exerts on Flt3L‐induced DC development has not been thoroughly examined. Here, we report that GM‐CSF alters Flt3L‐induced DC development. When BM cells were cultured with both Flt3L and GM‐CSF, few CD8+ equivalent DCs or plasmacytoid DCs developed compared to cultures supplemented with Flt3L alone. The disappearance of these two cell subsets in GM‐CSF + Flt3L culture was not a result of simple inhibition of their development, but a diversion of the original differentiation trajectory to form a new cell population. As a consequence, both DC progeny and their functions were altered. The effect of GM‐CSF on DC subset development was confirmed in vivo. First, the CD8+ DC numbers were increased under GM‐CSF deficiency (when either GM‐CSF or its receptor was ablated). Second, this population was decreased under GM‐CSF hyperexpression (by transgenesis or by Listeria infection). Our finding that GM‐CSF dominantly changes the regulation of DC development in vitro and in vivo has important implications for inflammatory diseases or GM‐CSF therapy.     

IL‐33 promotes DC development in BM culture by triggering GM‐CSF production

Short‐term DC cultures generated with GM‐CSF and other cytokines have markedly improved our ability to study the immunobiology of DC. Here, we tested 65 cytokines individually for their potential to promote the generation of CD11c⁺ cells in a murine BM culture system. In addition to several cytokines known to promote DC survival and/or growth, IL‐33 was found to augment DC development time‐ and dose‐dependently. Although the resulting CD11c⁺ cells generated in the presence of IL‐33 exhibited a typical dendritic morphology, they expressed MHC class II molecules only at modest levels, showed negligible responses to TLR ligands, produced no detectable IL‐12 p70, displayed PD‐L1 and PD‐L2 on the surface, and failed to activate immunologically naïve T cells efficiently. IL‐33‐induced expansion of CD11c⁺ cells was completely blocked by anti‐GM‐CSF mAb, and GM‐CSF mRNA and protein expression in BM culture was markedly elevated by added IL‐33, indicating that IL‐33 promotes in vitro DC generation indirectly by a GM‐CSF‐dependent manner. With regard to the cellular source, IL‐33‐dependent GM‐CSF production was observed exclusively within the CD45⁺/FcεRI⁺ BM population. Not only do our results reinforce the notion that GM‐CSF serves as a primary DC growth factor, but they also reveal a previously unrecognized mechanism supporting DC development.     

An optimized Protocol for Human M2 Macrophages using M‐CSF and IL‐4/IL‐10/TGF‐β Yields a Dominant Immunosuppressive Phenotype

Monocytes are highly abundant circulatory effector cells and play a vital role in driving or resolving inflammatory processes depending on their activation phenotype. We investigated and compared a panel of polarization protocols of blood‐derived monocytes to achieve a stable, optimal and effective regimen for in vitro induction of immunosuppressive human macrophages, evaluating their surface receptor expression, cytokine profile, scavenging function and ability to suppress T‐cell proliferation. Importantly, we assessed the effect of copolarization or secondary pro‐inflammatory stimulation of a primary anti‐inflammatory activation phenotype. A combination of IL‐4/IL‐10/TGF‐β yielded a relatively stable and dominant immunosuppressive phenotype characterized by higher IL‐10 production and down‐regulated TNF‐α, IL‐6, CD86, CD274 and MHC II expression. Functionally, IL‐4/IL‐10/TGF‐β‐stimulated macrophages (M2) had a potent deactivating effect on a subsequent pro‐inflammatory LPS/IFNγ‐activated macrophage (M1) stimulation and significantly suppressed T‐cell proliferation. Monocytes derived from patients with chronic inflammatory diseases could be induced to be anti‐inflammatory using this protocol. Pre‐differentiation with GM‐CSF or M‐CSF was further demonstrated to enhance final M1/M2 activation status. Our findings indicate a robust polarization protocol for generation of specific immunosuppressive human monocyte‐derived macrophages.     

Evaluation of peroxisome proliferator‐activated receptor agonists on interleukin‐5‐induced eosinophil differentiation

Peroxisome proliferator‐activated receptor (PPAR) agonists have been suggested as novel therapeutics for the treatment of inflammatory lung disease, such as allergic asthma. Treatment with PPAR agonists has been shown to inhibit airway eosinophilia in murine models of allergic asthma, which can occur through several mechanisms including attenuated generation of chemoattractants (e.g. eotaxin) and decreased eosinophil migrational responses. In addition, studies report that PPAR agonists can inhibit the differentiation of several cell types. To date, no studies have examined the effects of PPAR agonists on interleukin‐5 (IL‐5) ‐induced eosinophil differentiation from haemopoietic progenitor cells. Non‐adherent mononuclear cells or CD34⁺ cells isolated from the peripheral blood of allergic subjects were grown for 2 weeks in Methocult^® cultures with IL‐5 (10 ng/ml) and IL‐3 (25 ng/ml) in the presence of 1–1000 nm PPARα agonist (GW9578), PPARβ/δ agonist (GW501516), PPARγ agonist (rosiglitazone) or diluent. The number of eosinophil/basophil colony‐forming units (Eo/B CFU) was quantified by light microscopy. The signalling mechanism involved was assessed by phosphoflow. Blood‐extracted CD34⁺ cells cultured with IL‐5 or IL‐5 + IL‐3 formed Eo/B CFU, which were significantly inhibited by rosiglitazone (100 nm , P < 0·01) but not GW9578 or GW501516. In addition, rosglitazone significantly inhibited IL‐5‐induced phosphorylation of extracellular signal‐regulated kinase 1/2. We observed an inhibitory effect of rosiglitazone on eosinophil differentiation in vitro, mediated by attenuation of the extracellular signal‐regulated kinase 1/2 signalling pathway. These findings indicate that the PPARγ agonist can attenuate tissue eosinophilia by interfering with local differentiative responses.     

Urban particulate matter stimulation of human dendritic cells enhances priming of naive CD8 T lymphocytes

Epidemiological studies have consistently shown associations between elevated concentrations of urban particulate matter (UPM) air pollution and exacerbations of asthma and chronic obstructive pulmonary disease, which are both associated with viral respiratory infections. The effects of UPM on dendritic cell (DC) ‐stimulated CD4 T lymphocytes have been investigated previously, but little work has focused on CD8 T‐lymphocyte responses despite their importance in anti‐viral immunity. To address this, we examined the effects of UPM on DC‐stimulated naive CD8 T‐cell responses. Expression of the maturation/activation markers CD83, CCR7, CD40 and MHC class I on human myeloid DCs (mDCs) was characterized by flow cytometry after stimulation with UPMin vitro in the presence/absence of granulocyte–macrophage colony‐stimulating factor (GM‐CSF). The capacity of these mDCs to stimulate naive CD8 T‐lymphocyte responses in allogeneic co‐culture was then assessed by measuring T‐cell cytokine secretion using cytometric bead array, and proliferation and frequency of interferon‐γ (IFN‐γ)‐producing T lymphocytes by flow cytometry. Treatment of mDCs with UPM increased expression of CD83 and CCR7, but not MHC class I. In allogeneic co‐cultures, UPM treatment of mDCs enhanced CD8 T‐cell proliferation and the frequency of IFN‐γ⁺ cells. The secretion of tumour necrosis factor‐α, interleukin‐13, Granzyme A and Granzyme B were also increased. GM‐CSF alone, and in concert with UPM, enhanced many of these T‐cell functions. The PM‐induced increase in Granzyme A was confirmed in a human experimental diesel exposure study. These data demonstrate that UPM treatment of mDCs enhances priming of naive CD8 T lymphocytes and increases production of pro‐inflammatory cytokines. Such UPM‐induced stimulation of CD8 cells may potentiate T‐lymphocyte cytotoxic responses upon concurrent airway infection, increasing bystander damage to the airways.     

Cytokine‐dependent regulation of dendritic cell differentiation in the splenic microenvironment

The DC‐derived chemokine CCL17, a ligand of CCR4, has been shown to promote various inflammatory diseases such as atopic dermatitis, atherosclerosis, and inflammatory bowel disease. Under steady‐state conditions, and even after systemic stimulation with LPS, CCL17 is not expressed in resident splenic DCs as opposed to CD8α^?CD11b⁺ LN DCs, which produce large amounts of CCL17 in particular after maturation. Upon systemic NKT cell activation through α‐galactosylceramide stimulation however, CCL17 can be upregulated in both CD8α^? and CD8α⁺ splenic DC subsets and enhances cross‐presentation of exogenous antigens. Based on genome‐wide expression profiling, we now show that splenic CD11b⁺ DCs are susceptible to IFN‐γ‐mediated suppression of CCL17, whereas LN CD11b⁺CCL17⁺ DCs downregulate the IFN‐γR and are much less responsive to IFN‐γ. Under inflammatory conditions, particularly in the absence of IFN‐γ signaling in IFN‐γRKO mice, CCL17 expression is strongly induced in a major proportion of splenic DCs by the action of GM‐CSF in concert with IL‐4. Our findings demonstrate that the local cytokine milieu and differential cytokine responsiveness of DC subsets regulate lymphoid organ specific immune responses at the level of chemokine expression.     

CD46 accelerates macrophage‐mediated host susceptibility to meningococcal sepsis in a murine model

CD46, a membrane cofactor expressed on all nucleated human cells, plays an essential role in suppressing autoimmune reactions and protecting host cells from complement‐mediated attack. Human transgenic CD46 homozygous mice (CD46^+/+) are prone to lethal sepsis upon infection with Neisseria meningitidis (N. meningitidis). However, the underlying mechanisms are poorly understood. Here, we determined thatCD46^+/+ mice produce large numbers of M1 type macrophages with enhanced surface expression of MHC II and production of pro‐inflammatory mediators such as IL‐6, TNF, IL‐12, and IL‐1β In the presence of M‐CSF or GM‐CSF, CD46 signaling enhances monocyte‐macrophage differentiation. Additionally, CD46^+/+ macrophages rapidly undergo apoptosis upon LPS challenge or meningococcal infection, which could contribute to uncontrolled bacterial dissemination in vivo. Adoptive transfer of CD46^+/+ peritoneal macrophages aggravated septic responses in wild‐type mice, but the depletion of macrophages partially alleviated septic reactions in CD46^+/+ mice after N. meningitidis infection. Our findings reveal a novel role of CD46 in accelerating inflammatory responses upon meningococcal infection or LPS stimulation by regulating the functional polarization and survival of macrophages.     

Modulation of pulmonary DC function by vaccine‐encoded GM‐CSF enhances protective immunity against Mycobacterium tuberculosis infection

The rational design of new vaccines engineered to target key components of the host immune response is crucial to aid control of important infectious diseases such as tuberculosis. In this report, we determined whether modifying the function of pulmonary APC could improve protection against infection with Mycobacterium tuberculosis. Targeted delivery to the lung of the cytokine GM‐CSF, expressed by the Mycobacterium bovis BCG vaccine strain, increased pulmonary DC numbers and secretion of the immunoregulatory cytokine IL‐12, compared with parental BCG immunization. This impact on APC number by BCG:GM‐CSF resulted in accelerated priming of antigen‐specific CD4⁺ T cells in the mediastinal lymph nodes and increased migration of activated CD4⁺ T cells into the lung. i.n. administration of BCG:GM‐CSF resulted in significantly increased protection against M. tuberculosis infection compared with mice vaccinated with BCG alone. BCG:GM‐CSF exhibited an improved safety profile, as immunodeficient RAG1^?/? mice vaccinated i.n. with BCG:GM‐CSF survived significantly longer than control BCG‐vaccinated mice. These data demonstrate that manipulating immune cells in the lung by BCG‐based delivery of GM‐CSF can assist the development of protective mucosal immunity against pulmonary bacterial infection.     

Sca‐1+ cardiac fibroblasts promote development of heart failure

The causative effect of GM‐CSF produced by cardiac fibroblasts to development of heart failure has not been shown. We identified the pathological GM‐CSF‐producing cardiac fibroblast subset and the specific deletion of IL‐17A signaling to these cells attenuated cardiac inflammation and heart failure. We describe here the CD45^?CD31^?CD29⁺mEF‐SK4⁺PDGFRα⁺Sca‐1⁺periostin⁺ (Sca‐1⁺) cardiac fibroblast subset as the main GM‐CSF producer in both experimental autoimmune myocarditis and myocardial infarction mouse models. Specific ablation of IL‐17A signaling to Sca‐1⁺periostin⁺ cardiac fibroblasts (Postn^CreIl17ra^fl/fl) protected mice from post‐infarct heart failure and death. Moreover, Postn^CreIl17ra^fl/fl mice had significantly fewer GM‐CSF‐producing Sca‐1⁺ cardiac fibroblasts and inflammatory Ly6C^hi monocytes in the heart. Sca‐1⁺ cardiac fibroblasts were not only potent GM‐CSF producers, but also exhibited plasticity and switched their cytokine production profiles depending on local microenvironments. Moreover, we also found GM‐CSF‐positive cardiac fibroblasts in cardiac biopsy samples from heart failure patients of myocarditis or ischemic origin. Thus, this is the first identification of a pathological GM‐CSF‐producing cardiac fibroblast subset in human and mice hearts with myocarditis and ischemic cardiomyopathy. Sca‐1⁺ cardiac fibroblasts direct the type of immune cells infiltrating the heart during cardiac inflammation and drive the development of heart failure.     

Pathogenic IL‐23 signaling is required to initiate GM‐CSF‐driven autoimmune myocarditis in mice

Using a mouse model of experimental autoimmune myocarditis (EAM), we showed for the first time that IL‐23 stimulation of CD4⁺ T cells is required only briefly at the initiation of GM‐CFS‐dependent cardiac autoimmunity. IL‐23 signal, acting as a switch, turns on pathogenicity of CD4⁺ T cells, and becomes dispensable once autoreactivity is established. Il23a^?/? mice failed to mount an efficient Th17 response to immunization, and were protected from myocarditis. However, remarkably, transient IL‐23 stimulation ex vivo fully restored pathogenicity in otherwise nonpathogenic CD4⁺ T cells raised from Il23a^?/? donors. Thus, IL‐23 may no longer be necessary to uphold inflammation in established autoimmune diseases. In addition, we demonstrated that IL‐23‐induced GM‐CSF mediates the pathogenicity of CD4⁺ T cells in EAM. The neutralization of GM‐CSF abrogated cardiac inflammation. However, sustained IL‐23 signaling is required to maintain IL‐17A production in CD4⁺ T cells. Despite inducing inflammation in Il23a^?/? recipients comparable to wild‐type (WT), autoreactive CD4⁺ T cells downregulated IL‐17A production without persistent IL‐23 signaling. This divergence on the controls of GM‐CSF‐dependent pathogenicity on one side and IL‐17A production on the other side may contribute to the discrepant efficacies of anti‐IL‐23 therapy in different autoimmune diseases.     

Hepatic B cells are readily activated by Toll‐like receptor‐4 ligation and secrete less interleukin‐10 than lymphoid tissue B cells

B cells perform various immunological functions that include production of antibody, presentation of antigens, secretion of multiple cytokines and regulation of immune responses mainly via their secretion of interleukin (IL)‐10. While the liver is regarded both as an important immune organ and a tolerogenic environment, little is known about the functional biology of hepatic B cells. In this study we demonstrate that, following lipopolysaccharide (LPS) stimulation in vivo, normal mouse hepatic B cells rapidly increase their surface expression of CD39, CD40, CD80 and CD86, and produce significantly elevated levels of proinflammatory interferon (IFN)‐γ, IL‐6 and tumour necrosis factor (TNF)‐α compared with splenic B cells. Moreover, LPS‐activated hepatic B cells produce very low levels of IL‐10 compared with activated splenic B cells that produce comparatively high levels of this immunosuppressive cytokine. Splenic, but not hepatic, B cells inhibited the activation of liver conventional myeloid dendritic cells (mDCs). Furthermore, compared with the spleen, the liver exhibited significantly smaller proportions of B1a and marginal zone‐like B cells, which have been shown to produce IL‐10 upon LPS stimulation. These data suggest that, unlike in the spleen, IL‐10‐producing regulatory B cells in the liver are not a prominent cell type. Consistent with this, when compared with liver conventional mDCs from B cell‐deficient mice, those from B cell‐competent wild‐type mice displayed enhanced expression of the cell surface co‐stimulatory molecule CD86, greater production of proinflammatory cytokines (IFN‐γ, IL‐6, IL‐12p40) and reduced secretion of IL‐10. These findings suggest that hepatic B cells have the potential to initiate rather than regulate inflammatory responses.     

The generation of human innate lymphoid cells is influenced by the source of hematopoietic stem cells and by the use of G‐CSF

NK cells play a central role in the haploidentical HSC transplantation (HSCT) to cure high‐risk leukemias. Other innate lymphoid cells (ILCs) have been proposed to exert a protective role in graft‐versus‐host disease and could also contribute to anti‐microbial defence and to lymphoid tissue remodeling. Thus, we investigated the ILC differentiation potential of HSCs isolated from BM, mobilized peripheral blood (PB), and umbilical cord blood (UCB). BM CD34⁺ cells are enriched in lymphoid‐committed precursors, while PB CD34⁺ cells preferentially contain myeloid precursors. In vitro differentiation experiments revealed that the highest and the lowest CD56⁺CD161⁺ ILC recovery was detected in UCB and PB HSC cultures, respectively. Among CD56⁺CD161⁺ ILCs, the ratio between NK cells and ILC3s was similar for all HSC analyzed. ILC recovery in PB CD34⁺ cultures was lower for G‐CSF‐mobilized HSCs (good mobilizers) than for G‐CSF+plerixafor‐mobilized HSC (poor mobilizers). Moreover, G‐CSF inhibited in vitro ILC recovery and the degree of inhibition was proportional to the time of exposure to the cytokine. Thus, although all common sources of HSC for transplant differentiate towards ILCs, substantial differences exist among different sources and G‐CSF may influence ILC recovery. These data offer new clues for a better understanding of the immune reconstitution after HSCT.     

Engulfment of Hb‐activated platelets differentiates monocytes into pro‐inflammatory macrophages in PNH patients

The distinct response shown by different phenotypes of macrophages and monocytes under various clinical conditions has put the heterogeneity of these cells into focus of investigation for several diseases. Recently, we have described that after engulfing hemoglobin (Hb)‐activated platelets, classical monocytes differentiated into pro‐inflammatory phenotypes, which were abundant in the circulation of paroxysmal nocturnal hemoglobinuria (PNH) and sickle cell disease patients. Our current study shows that upon engulfment of Hb‐activated platelets, monocytes differentiate into M1‐macrophages under M1‐polarization stimulus (GM‐CSF, IFN‐γ + LPS). When grown under M2‐polarization stimulus (M‐CSF, IL‐4 + IL13), the cells exhibited an M1‐like phenotype, secreted elevated levels of pro‐inflammatory cytokines including TNF‐α and IL‐1β, and displayed loss of the secretion of cytokine such as IL‐10 and also phagocytic ability unlike the conventional M2 macrophages. Interestingly, when differentiated under the above polarization stimulus, monocytes from PNH patients expressed high levels of CD80 and phospho‐STAT1, like M1 macrophages. Hemolytic mice also exhibited a gradual increase in monocyte–platelet aggregates in circulation and accumulation of CD80^high macrophages in thioglycollate‐induced inflamed peritoneum. The spleen of the mice was also populated by CD80^high macrophages with compromised phagocytic capacity. Our findings suggest that the hemolytic environment and specifically the Hb‐activated platelets, which are abundant in circulation during intravascular hemolysis, closely regulate monocyte differentiation.     

Gr‐1 antibody induces STAT signaling,macrophage marker expression and abrogation of myeloid‐derived suppressor cell activity in BM cells

The Gr‐1 (RB6‐8C5) Ab binds with high affinity to mouse Ly‐6G molecules and to a lower extent to Ly‐6C and has been widely used for cell depletion in infected or tumor‐bearing mice. Here we found that Gr‐1 treatment of BM cells in vitro and in vivo showed no depleting effects. The epitope recognized by the Gr‐1 Ab overlapped with Ly‐6G (1A8 Ab) but not Ly‐6C (ER‐MP20 Ab). In vitro the Gr‐1 Ab transmitted signals via STAT‐1, STAT‐3 and STAT‐5 into BM cells, similar to GM‐CSF. In healthy mice injected with the Gr‐1 Ab, the Ab remained attached to the surface of myeloid cells for at least four days. Gr‐1 Ab induced myeloid cell expansion, upregulation of macrophage markers, but not the DC marker CD11c. Suppressor activity of two distinct Gr‐1^high and Gr‐1^low expressing BM‐myeloid‐derived suppressor cell subsets was transiently ablated by Gr‐1 Ab injection. Depleting effects of Gr‐1 Ab could only be observed on inflammatory Ly‐6C^intLy‐6G^high neutrophils from the peritoneal cavity, which occurred via apoptosis and was associated with the absence of Mcl‐1 expression. Together, Gr‐1 Ab induces signals leading to myelopoiesis and affects myeloid‐derived suppressor cell activity, suggesting functional roles for Ly‐6C/G molecules in macrophage differentiation and neutrophil apoptosis.     

Exposure to inflammatory cytokines selectively limits GM‐CSF production by induced T regulatory cells

Interest in manipulating the immunosuppressive powers of Foxp3‐expressing T regulatory cells as an immunotherapy has been tempered by their reported ability to produce proinflammatory cytokines when manipulated in vitro, or in vivo. Understanding processes that can limit this potentially deleterious effect of Treg cells in a therapeutic setting is therefore important. Here, we have studied this using induced (i) Treg cells in which de novo Foxp3 expression is driven by TCR‐stimulation in vitro in the presence of TGF‐β. We show that iTreg cells can produce significant amounts of three proinflammatory cytokines (IFN‐γ, GM‐CSF and TNF‐α) upon secondary TCR stimulation. GM‐CSF is a critical T‐cell derived cytokine for the induction of EAE in mice. Despite their apparent capacity to produce GM‐CSF, myelin autoantigen‐responsive iTreg cells were unable to provoke EAE. Instead, they maintained strong suppressive function in vivo, preventing EAE induction by their CD4⁺Foxp3^? counterparts. We identified that although iTreg cells maintained the ability to produce IFN‐γ and TNF‐α in vivo, their ability to produce GM‐CSF was selectively degraded upon antigen stimulation under inflammatory conditions. Furthermore, we show that IL‐6 and IL‐27 individually, or IL‐2 and TGF‐β in combination, can mediate the selective loss of GM‐CSF production by iTreg cells.     

The atypical IκB protein IκBNS is important for Toll‐like receptor‐induced interleukin‐10 production in B cells

Although a major function of B cells is to mediate humoral immunity by producing antigen‐specific antibodies, a specific subset of B cells is important for immune suppression, which is mainly mediated by the secretion of the anti‐inflammatory cytokine interleukin‐10 (IL‐10). However, the mechanism by which IL‐10 is induced in B cells has not been fully elucidated. Here, we report that IκB_NS, an inducible nuclear IκB protein, is important for Toll‐like receptor (TLR)‐mediated IL‐10 production in B cells. Studies using IκB_NS knockout mice revealed that the number of IL‐10‐producing B cells is reduced in IκB_NS^?/? spleens and that the TLR‐mediated induction of cytoplasmic IL‐10‐positive cells and IL‐10 secretion in B cells are impaired in the absence of IκB_NS. The impairment of IL‐10 production by a lack of IκB_NS was not observed in TLR‐triggered macrophages or T‐cell‐receptor‐stimulated CD4⁺ CD25⁺ T cells. In addition, IκB_NS‐deficient B cells showed reduced expression of Prdm1 and Irf4 and failed to generate IL‐10⁺ CD138⁺ plasmablasts. These results suggest that IκB_NS is selectively required for IL‐10 production in B cells responding to TLR signals, so defining an additional role for IκB_NS in the control of the B‐cell‐mediated immune responses.