CD8+ T cells producing IL‐3 and IL‐5 in non‐IgE‐mediated eosinophilic diseases

The cytokines IL‐5, IL‐3, and GM‐CSF are crucial for eosinophil development, survival, and function. To better understand their role in non‐IgE‐mediated eosinophilic diseases, we investigated plasma levels of these cytokines as well as cytokine expression in peripheral blood T cells. While we did not find any evidence for an involvement of T‐cell‐derived GM‐CSF, some of these patients did show an increased proportion of IL‐5‐ or IL‐3‐producing CD4⁺ T cells. However, in a significant proportion of patients, IL‐5‐producing CD8⁺ T cells, so‐called Tc2 cells, which in healthy donors can only be detected at very low levels, were prominent. Furthermore, increased IL‐3 production by CD8⁺ T cells was also observed, strongly supporting the notion that CD8⁺ T cells, not just CD4⁺ T cells, must also be considered as a potential source of the cytokines promoting eosinophilia.     

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.     

IL‐33 broadens its repertoire to affect DC

IL‐33 is a novel multi‐functional IL‐1 family member that, in contrast to other family members, is associated with Th2 responses. IL‐33 signals via a heterodimer composed of its receptor, IL‐1 receptor‐like‐1 (IL‐1RL1), more commonly known as ST2L, and the IL‐1R accessory protein. ST2L is expressed by endothelial cells, mast cells, basophils, Th2 cells, and DC. IL‐33 has been associated with several immune‐mediated disorders, including asthma, arthritis, and inflammatory bowel disease. In contrast, there is evidence that IL‐33 can inhibit atherosclerosis development. A report in this issue of the European Journal of Immunology reveals a novel function of IL‐33: the ability to promote myeloid DC generation in murine BM cell cultures, by triggering GM‐CSF production by other BM cells, likely basophils. DC generated in the presence of IL‐33 are maturation resistant, with only minimal T‐cell stimulatory ability, associated with comparatively high levels of programmed death receptor ligand expression. This commentary discusses several questions raised by these findings, and provides a basis for further evaluation of IL‐33 and ST2L in regulation of APC generation and function in both innate and adaptive immunity.     

GM‐CSF therapy inhibits chronic graft‐versus‐host disease via expansion of regulatory T cells

Regulatory T cells (Tregs) attenuate excessive immune responses, making their expansion beneficial in immune‐mediated diseases, including allogeneic bone marrow transplantation associated with graft‐versus‐host disease (GVHD). In addition to interleukin‐2, Tregs require T‐cell receptor and costimulatory signals from antigen‐presenting cells, such as DCs, for their optimal proliferation. Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) increases DC number and may promote DC‐dependent Treg proliferation. Here, we demonstrate that GM‐CSF treatment increases CD4⁺CD8^– DCs, which are associated with Treg expansion. In a mouse model of chronic GVHD (cGVHD), GM‐CSF therapy expanded Tregs, protected against the development of skin GVHD, and regulated both Th1 and Th17 responses in the peripheral lymph nodes, resulting in an attenuation of skin cGVHD. Notably, the expanded Tregs were instrumental to GM‐CSF‐mediated cGVHD inhibition, which was dependent upon an increased ratio of Tregs to conventional T cells rather than augmentation of suppressive function. These data suggest that GM‐CSF induces Treg proliferation by expanding CD4⁺CD8^? DCs, which in turn regulate alloimmune responses in a cGVHD mouse model. Thus, GM‐CSF could be used as a therapeutic DC modulator to induce Treg expansion and to inhibit excessive alloimmune responses in immune‐related diseases.     

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.     

BDCA3 expression is associated with high IFN‐λ production by CD34+‐derived dendritic cells generated in the presence of GM‐CSF,IL‐4, and/or TGF‐β

High BDCA3 expression is associated with a specific human IFN‐λ‐producing dendritic cell (DC) subset. However, BDCA3 has also been detected on other DC subsets. Thus far, development and function of BDCA3 expression on DCs remains poorly understood. Human Langerhans cells (LCs) and interstitial DCs (intDCs) can be generated in vitro by differentiation of CD34⁺ hematopoietic progenitors via distinct precursor DCs (preDCs), CD1a⁺ preDCs, and CD14⁺ preDCs, respectively. Here, we identified BDCA3 expression in this well‐known GM‐CSF/TNF‐α‐driven culture system and described the effect of IL‐4 and/or TGF‐β on induction of BDCA3 expression. In control or TGF‐β cultures, BDCA3 was only detected on CD14⁺ preDC‐derived intDCs. IL‐4 induced BDCA3 expression in both CD14⁺‐derived and CD1a⁺‐derived cultures. TGF‐β and IL‐4 together further increased CD14⁺‐derived and CD1a⁺‐derived BDCA3⁺ DC frequencies, which partly expressed CLEC9A, but were not identical to the BDCA3^highCLEC9A⁺ DC subset in vivo. Importantly, BDCA3⁺ cells, but not BDCA3^? cells, in this system produced high IFN‐λ levels upon polyinosinic:polycytidylic acid (polyI:C) stimulation. This culture system, in which BDCA3 expression is preferentially associated with the intDC lineage and IFN‐λ‐producing capacity, will greatly contribute to further research on the function and regulation of BDCA3 expression and IFN‐λ production by DCs.     

Type I IFN regulate DC turnover in vivo

DC are the most potent antigen‐presenting cells that recognise signs of infection and serve as the main activators of naïve T cells. We have previously shown that type I IFN (IFN‐I) are produced by DC and can act in an autocrine manner to activate DC. In the present study, we have investigated the role of IFN‐I in regulating the turnover and lifespan of DC. We found that DC, especially the CD8α⁺ subset, from type I IFN receptor knock out (IFNAR KO) mice, display a reduced turnover rate when compared with DC from WT mice, as revealed by BrdU labelling kinetics. In vitro, IFNAR KO BM precursor cells cultured in the presence of GM‐CSF generated CD11c⁺ DC less efficiently than WT BM, and the IFNAR KO DC that arose displayed reduced migratory ability. Interestingly, splenic DC from IFNAR KO mice exhibited a higher survival rate in short‐term culture compared with control DC. Exposure to IFN‐I in vivo markedly increased the turnover rate of splenic DC, particularly CD8α⁺ DC, which was preceded by a transient induction of apoptosis. In accordance with this, IFN‐I stimulated the apoptosis of splenic DC in vitro. Overall, our data indicate that IFN‐I are important regulators of DC turnover in vivo and suggest that these cytokines may exert this function through the modulation of multiple processes involving DC apoptosis, proliferation and migration.     

Depletion of CD11c+ cells in the CD11c.DTR model drives expansion of unique CD64+ Ly6C+ monocytes that are poised to release TNF‐α

Dendritic cells (DCs) play a vital role in innate and adaptive immunities. Inducible depletion of CD11c⁺ DCs engineered to express a high‐affinity diphtheria toxin receptor has been a powerful tool to dissect DC function in vivo. However, despite reports showing that loss of DCs induces transient monocytosis, the monocyte population that emerges and the potential impact of monocytes on studies of DC function have not been investigated. We found that depletion of CD11c⁺ cells from CD11c.DTR mice induced the expansion of a variant CD64⁺ Ly6C⁺ monocyte population in the spleen and blood that was distinct from conventional monocytes. Expansion of CD64⁺ Ly6C⁺ monocytes was independent of mobilization from the BM via CCR2 but required the cytokine, G‐CSF. Indeed, this population was also expanded upon exposure to exogenous G‐CSF in the absence of DC depletion. CD64⁺ Ly6C⁺ monocytes were characterized by upregulation of innate signaling apparatus despite the absence of inflammation, and an increased capacity to produce TNF‐α following LPS stimulation. Thus, depletion of CD11c⁺ cells induces expansion of a unique CD64⁺ Ly6C⁺ monocyte population poised to synthesize TNF‐α. This finding will require consideration in experiments using depletion strategies to test the role of CD11c⁺ DCs in immunity.     

Hierarchy of immunosuppressive strength among myeloid‐derived suppressor cell subsets is determined by GM‐CSF

CD11b⁺/Gr‐1⁺ myeloid‐derived suppressor cells (MDSC) contribute to tumor immune evasion by restraining the activity of CD8⁺ T‐cells. Two major MDSC subsets were recently shown to play an equal role in MDSC‐induced immune dysfunctions: monocytic‐ and granulocytic‐like. We isolated three fractions of MDSC, i.e. CD11b⁺/Gr‐1^high, CD11b⁺/Gr‐1^int, and CD11b⁺/Gr‐1^low populations that were characterized morphologically, phenotypically and functionally in different tumor models. In vitro assays showed that CD11b⁺/Gr‐1^int cell subset, mainly comprising monocytes and myeloid precursors, was always capable to suppress CD8⁺ T‐cell activation, while CD11b⁺/Gr‐1^high cells, mostly granulocytes, exerted appreciable suppression only in some tumor models and when present in high numbers. The CD11b⁺/Gr‐1^int but not CD11b⁺/Gr‐1^high cells were also immunosuppressive in vivo following adoptive transfer. CD11b⁺/Gr‐1^low cells retained the immunosuppressive potential in most tumor models. Gene silencing experiments indicated that GM‐CSF was necessary to induce preferential expansion of both CD11b⁺/Gr‐1^int and CD11b⁺/Gr‐1^low subsets in the spleen of tumor‐bearing mice and mediate tumor‐induced tolerance whereas G‐CSF, which preferentially expanded CD11b⁺/Gr‐1^high cells, did not create such immunosuppressive environment. GM‐CSF also acted on granulocyte–macrophage progenitors in the bone marrow inducing local expansion of CD11b⁺/Gr‐1^low cells. These data unveil a hierarchy of immunoregulatory activity among MDSC subsets that is controlled by tumor‐released GM‐CSF.     

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.     

Modified vaccinia Ankara‐expressing Ag85A,a novel tuberculosis vaccine,is safe in adolescents and children,and induces polyfunctional CD4+ T cells

Modified vaccinia Ankara‐expressing Ag85A (MVA85A) is a new tuberculosis (TB) vaccine aimed at enhancing immunity induced by BCG. We investigated the safety and immunogenicity of MVA85A in healthy adolescents and children from a TB endemic region, who received BCG at birth. Twelve adolescents and 24 children were vaccinated and followed up for 12 or 6 months, respectively. Adverse events were documented and vaccine‐induced immune responses assessed by IFN‐γ ELISpot and intracellular cytokine staining. The vaccine was well tolerated and there were no vaccine‐related serious adverse events. MVA85A induced potent and durable T‐cell responses. Multiple CD4⁺ T‐cell subsets, based on expression of IFN‐γ, TNF‐α, IL‐2, IL‐17 and GM‐CSF, were induced. Polyfunctional CD4⁺ T cells co‐expressing IFN‐γ, TNF‐α and IL‐2 dominated the response in both age groups. A novel CD4⁺ cell subset co‐expressing these three Th1 cytokines and IL‐17 was induced in adolescents, while a novel CD4⁺ T‐cell subset co‐expressing Th1 cytokines and GM‐CSF was induced in children. Ag‐specific CD8⁺ T cells were not detected. We conclude that in adolescents and children MVA85A safely induces the type of immunity thought to be important in protection against TB. This includes induction of novel Th1‐cell populations that have not been previously described in humans.     

Different interleukin‐17‐secreting Toll‐like receptor+ T‐cell subsets are associated with disease activity in multiple sclerosis

Signalling through Toll‐like receptors (TLRs) may play a role in the pathogenesis of autoimmune diseases, such as multiple sclerosis (MS). In the present study, the expression of TLR‐2, ‐4 and ‐9 was significantly higher on CD4⁺ and CD8⁺ T‐cells from MS patients compared to healthy individuals. Following in‐vitro activation, the proportion of interleukin (IL)‐17⁺ and IL‐6⁺ CD4⁺ and CD8⁺ T‐cells was higher in the patients. In addition, the proportion of IFN‐γ‐secreting TLR⁺ CD8⁺ T‐cells was increased in MS patients. Among different IL‐17⁺ T‐cell phenotypes, the proportion of IL‐17⁺ TLR⁺ CD4⁺ and CD8⁺ T‐cells producing IFN‐γ or IL‐6 were positively associated with the number of active brain lesions and neurological disabilities. Interestingly, activation of purified CD4⁺ and CD8⁺ T‐cells with ligands for TLR‐2 (Pam3Csk4), TLR‐4 [lipopolysaccharide (LPS)] and TLR‐9 [oligodeoxynucleotide (ODN)] directly induced cytokine production in MS patients. Among the pathogen‐associated molecular patterns (PAMPs), Pam3Csk4 was more potent than other TLR ligands in inducing the production of all proinflammatory cytokines. Furthermore, IL‐6, IFN‐γ, IL‐17 and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) levels produced by Pam3Csk4‐activated CD4⁺ cells were directly associated with disease activity. A similar correlation was observed with regard to IL‐17 levels released by Pam3Csk4‐stimulated CD8⁺ T‐cells and clinical parameters. In conclusion, our data suggest that the expansion of different T helper type 17 (Th17) phenotypes expressing TLR‐2, ‐4 and ‐9 is associated with MS disease activity, and reveals a preferential ability of TLR‐2 ligand in directly inducing the production of cytokines related to brains lesions and neurological disabilities.     

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.     

The transduction pattern of IL‐12‐encoding lentiviral vectors shapes the immunological outcome

In situ modification of antigen‐presenting cells garnered interest in cancer immunotherapy. Therefore, we developed APC‐targeted lentiviral vectors (LVs). Unexpectedly, these LVs were inferior vaccines to broad tropism LVs. Since IL‐12 is a potent mediator of antitumor immunity, we evaluated whether this proinflammatory cytokine could enhance antitumor immunity of an APC‐targeted LV‐based vaccine. Therefore, we compared subcutaneous administration of broad tropism LVs (VSV‐G‐LV) with APC‐targeted LVs (DC2.1‐LV)‐encoding enhanced GFP and ovalbumin, or IL‐12 and ovalbumin in mice. We show that codelivery of IL‐12 by VSV‐G‐LVs or DC2.1‐LVs augments CD4⁺ or CD8⁺ T‐cell proliferation, respectively. Furthermore, we demonstrate that codelivery of IL‐12 enhances the CD4⁺ T_H1 profile irrespective of its delivery mode, while an increase in cytotoxic and therapeutic CD8⁺ T cells was only induced upon VSV‐G‐LV injection. While codelivery of IL‐12 by DC2.1‐LVs did not enhance CD8⁺ T‐cell performance, it increased expression of inhibitory checkpoint markers Lag3, Tim3, and PD‐1. Finally, the discrepancy between CD4⁺ T‐cell stimulation with and without functional CD8⁺ T‐cell stimulation by VSV‐G‐ and DC2.1‐LVs is partly explained by the observation that IL‐12 relieves CD8⁺ T cells from CD4⁺ T‐cell help, implying that a T_H1 profile is of minor importance for antitumor immunotherapy if IL‐12 is exogenously delivered.     

IL‐10 promotes homeostatic proliferation of human CD8+ memory T cells and,when produced by CD1c+ DCs,shapes naive CD8+ T‐cell priming

IL‐10 is an anti‐inflammatory cytokine that inhibits maturation and cytokine production of dendritic cells (DCs). Although mature DCs have the unique capacity to prime CD8⁺ CTL, IL‐10 can promote CTL responses. To understand these paradoxic findings, we analyzed the role of IL‐10 produced by human APC subsets in T‐cell responses. IL‐10 production was restricted to CD1c⁺ DCs and CD14⁺ monocytes. Interestingly, it was differentially regulated, since R848 induced IL‐10 in DCs, but inhibited IL‐10 in monocytes. Autocrine IL‐10 had only a weak inhibitory effect on DC maturation, cytokine production, and CTL priming with high‐affinity peptides. Nevertheless, it completely blocked cross‐priming and priming with low‐affinity peptides of a self/tumor‐antigen. IL‐10 also inhibited CD1c⁺ DC‐induced CD4⁺ T‐cell priming and enhanced Foxp3 induction, but was insufficient to induce T‐cell IL‐10 production. CD1c⁺ DC‐derived IL‐10 had also no effect on DC‐induced secondary expansions of memory CTL. However, IL‐15‐driven, TCR‐independent proliferation of memory CTL was enhanced by IL‐10. We conclude that DC‐derived IL‐10 selects high‐affinity CTL upon priming. Moreover, IL‐10 preserves established CTL memory by enhancing IL‐15‐dependent homeostatic proliferation. These combined effects on CTL priming and memory maintenance provide a plausible mechanism how IL‐10 promotes CTL responses in humans.     

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.     

Toll‐like receptor‐mediated eosinophil–basophil differentiation: autocrine signalling by granulocyte–macrophage colony‐stimulating factor in cord blood haematopoietic progenitors

Eosinophils are multi‐functional leucocytes that play a role in inflammatory processes including allergy and infection. Although bone marrow (BM) inflammatory cells are the main source of eosinophil‐basophil (Eo/B) differentiation‐inducing cytokines, a recent role has been demonstrated for cytokine induction through Toll‐like receptor (TLR)‐mediated signalling in BM progenitors. Having previously demonstrated that cord blood (CB) progenitors induce Eo/B colony‐forming units (CFU) after lipopolysaccharide (LPS) stimulation, we sought to investigate the intracellular mechanisms by which LPS induces Eo/B differentiation. Freshly isolated CD34‐enriched human CB cells were stimulated with LPS (and/or pharmacological inhibitors) and assessed for alterations in haematopoietic cytokine receptor expression and signalling pathways by flow cytometry, Eo/B CFU in methylcellulose cultures, and cytokine secretion using Luminex assays. The LPS stimulation resulted in a significant increase in granulocyte–macrophage colony‐stimulating factor (GM‐CSF)‐responsive, as opposed to interleukin‐5‐responsive, Eo/B CFU, which also correlated with significant increases in CD34⁺ cell GM‐CSFRα expression. Functionally, CB CD34⁺ cells secrete abundant amounts of GM‐CSF following LPS stimulation, via a p38 mitogen‐activated protein kinase (MAPK)‐dependent mechanism; this secretion was responsible for Eo/B CFU formation ex vivo, as shown by antibody blockade. We show for the first time that LPS stimulation of CB progenitor cells results in autocrine activation of p38 MAPK‐dependent GM‐CSF secretion facilitating Eo/B differentiation ex vivo. This work provides evidence that early life exposure to products of bacterial agents can modulate Eo/B differentiation, representing a novel mechanism by which progenitor cells can respond to microbial stimuli and so affect immune and inflammatory responses.     

GM‐CSF and IL‐4 are not involved in IVIG‐mediated amelioration of ITP in mice: a role for IL‐11 cannot be ruled out

Previously, we have reported that interleukin (IL)‐4, granulocyte–macrophage colony‐stimulating factor (GM‐CSF), and IL‐11, but not IL‐33, are up‐regulated in two strains of mice with immune thrombocytopenia (ITP) that are responsive to intravenous immunoglobulin (IVIg) treatment. Previously, IL‐4 was ruled out in the mechanism of IVIg; however, other publications have suggested this cytokine as a major player in the mechanism of IVIg action. Thus, we sought to further investigate a role for IL‐4 and, in addition, GM‐CSF and IL‐11 in the mechanism of action of IVIg using a murine model of ITP. A passive platelet antibody model was used to generate ITP in IL‐4 receptor knock‐out (IL‐4R^–/–), IL‐11 receptor knock‐out (IL‐11Rα^–/–) and GM‐CSF knock‐out (Csf2^–/–) mice. We also used a neutralizing antibody to IL‐11 and recombinant human IL‐11 (rhIL‐11) in addition to depleting basophils in vivo to study the effect of IVIg to ameliorate ITP. Our results showed that basophils, IL‐4 and GM‐CSF were unimportant in both ITP induction and its amelioration by IVIg. The role of IL‐11 in these processes was less clear. Even though IL‐11Rα^–/– mice with ITP responded to IVIg similarly to wild‐type (WT) mice, treatment of ITP WT mice with rhIL‐11 instead of IVIg showed an increase in platelet numbers and WT mice administered anti‐IL‐11 showed a significant reduction in the ability of IVIg to ameliorate the ITP. Our findings indicate that neither IL‐4, basophils or GM‐CSF have roles in IVIg amelioration of ITP; however, a role for IL‐11 requires further study.     

In vivo depletion of DC impairs the anti‐tumor effect of agonistic anti‐CD137 mAb

Anti‐CD137 mAb are capable of inducing tumor rejection in several syngeneic murine tumor models and are undergoing clinical trials for cancer. The anti‐tumor effect involves co‐stimulation of tumor‐specific CD8⁺ T cells. Whether antigen cross‐presenting DC are required for the efficacy of anti‐CD137 mAb treatment has never been examined. Here we show that the administration of anti‐CD137 mAb eradicates EG7‐OVA tumors by a strictly CD8β⁺ T‐cell‐dependent mechanism that correlates with increased CTL activity. Ex vivo analyses to determine the identity of the draining lymph node cell type responsible for tumor antigen cross‐presentation revealed that CD11c⁺ cells, most likely DC, are the main players in this tumor model. A minute number of tumor cells, revealed by the presence of OVA cDNA, reach tumor‐draining lymph nodes. Direct antigen presentation by tumor cells themselves also participates in anti‐OVA CTL induction. Using CD11c diphtheria toxin receptor‐green fluorescent protein→C57BL/6 BM chimeric mice, which allow for sustained ablation of DC with diphtheria toxin, we confirmed the involvement of DC in tumor antigen cross‐presentation in CTL induction against OVA_257–264 epitope and in the antitumor efficacy induced by anti‐CD137 mAb.