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Remerciements ECOFECT; International audience; The inflammasome is a signaling platform that is central to the innate immune responses to bacterial infections. Francisella tularensis is a bacterium replicating within the host cytosol. During F. tularensis subspecies novicida infection, AIM2, an inflammasome receptor sensing cytosolic DNA, activates caspase-1 in an ASC-dependent manner, leading to both pyroptosis and release of the proinflammatory cytokines IL-1β and IL-18. Activation of this canonical inflammasome pathway is key to limit F. novicida infection. In this study, by comparing the immune responses of AIM2 knockout (KO), ASC(KO), and Casp1(KO) mice in response to F. novicida infection, we observed that IFN-γ levels in the serum of Casp1(KO) mice were much higher than the levels observed in AIM2(KO) and ASC(KO) mice. This difference in IFN-γ production was due to a large production of IFN-γ by NK cells in Casp1(KO) mice that was not observed in ASC(KO) mice. The deficit in IFN-γ production observed in ASC(KO) mice was not due to a reduced Dock2 expression or to an intrinsic defect of ASC(KO) NK cells. We demonstrate that in infected Casp1(KO) mice, IFN-γ production is due to an ASC-dependent caspase-1-independent pathway generating IL-18. Furthermore, we present in vitro data suggesting that the recently described AIM2/ASC/caspase-8 noncanonical pathway is responsible for the caspase-1-independent IL-18 releasing activity. To our knowledge, this study is the first in vivo evidence of an alternative pathway able to generate in a caspase-1-independent pathway bioactive IL-18 to boost the production of IFN-γ, a cytokine critical for the host antibacterial response.

Erratum in J Immunol. 2013 Feb 1;190(3):1381. Daëron, Marc [added].; International audience; mAb therapy for experimental metastatic melanoma relies on activating receptors for the Fc portion of IgG (FcγR). Opposing results on the respective contribution of mouse FcγRI, FcγRIII, and FcγRIV have been reported using the gp75-expressing B16 melanoma and the protective anti-gp75 mAb TA99. We analyzed the contribution of FcγRs to this therapy model using bioluminescent measurement of lung metastases loads, novel mouse strains, and anti-FcγR blocking mAbs. We found that the TA99 mAb-mediated effects in a combination therapy using cyclophosphamide relied on activating FcγRs. The combination therapy, however, was not more efficient than mAb therapy alone. We demonstrate that FcγRI and, unexpectedly, FcγRIII contributed to TA99 mAb therapeutic effects, whereas FcγRIV did not. Therefore, FcγRIII and FcγRI are, together, responsible for anti-gp75 mAb therapy of B16 lung metastases. Our finding that mouse FcγRIII contributes to Ab-induced tumor reduction correlates with clinical data on its human functional equivalent human FcγRIIIA (CD16A).

CD3-specific Ab therapy results in a transient, self-limiting, cytokine-associated, flu-like syndrome in experimental animals and in patients, but the underlying mechanism for this spontaneous resolution remains elusive. By using an in vivo model of CD3-specific Ab-induced flu-like syndrome, we show in this paper that a single injection of sublethal dose of the Ab killed all TGF-β1−/− mice. The death of TGF-β1−/− mice was associated with occurrence of this uncontrolled flu-like syndrome, as demonstrated by a sustained storm of systemic inflammatory TNF and IFN-γ cytokines. We present evidence that deficiency of professional phagocytes to produce TGF-β1 after apoptotic T cell clearance may be responsible, together with hypersensitivity of T cells to both activation and apoptosis, for the uncontrolled inflammation. These findings indicate a key role for TGF-β1 and phagocytes in protecting the recipients from lethal inflammation and resolving the flu-like syndrome after CD3-specific Ab treatment. The study may also provide a novel molecular mechanism explaining the early death in TGF-β1−/− mice.

Sepsis is a systemic inflammatory response resulting from the inability of the host to contain the infection locally. Previously, we demonstrated that during severe sepsis there is a marked failure of neutrophil migration to the infection site, which contributes to dissemination of infection, resulting in high mortality. IL-17 plays an important role in neutrophil recruitment. Herein, we investigated the role of IL-17R signaling in polymicrobial sepsis induced by cecal ligation and puncture (CLP). It was observed that IL-17R-deficient mice, subjected to CLP-induced non-severe sepsis, show reduced neutrophil recruitment into the peritoneal cavity, spread of infection, and increased systemic inflammatory response as compared with C57BL/6 littermates. As a consequence, the mice showed an increased mortality rate. The ability of IL-17 to induce neutrophil migration was demonstrated in vivo and in vitro. Beside its role in neutrophil recruitment to the infection focus, IL-17 enhanced the microbicidal activity of the migrating neutrophils by a mechanism dependent on NO. Therefore, IL-17 plays a critical role in host protection during polymicrobial sepsis.

Several studies have reported that regulatory elements located 3′ of the IgH locus (namely hs3a, hs1,2, hs3b, and hs4) might play a role during class switch recombination (CSR) and Ig synthesis. While individual deletion of hs3a or hs1,2 had no effect, pairwise deletion of hs3b (an inverted copy of hs3a) and hs4 markedly affected CSR and Ig expression. Among these two elements, hs4 was tentatively presented with the master role due to its unique status within the 3′ regulatory region: distal position outside repeated regions, early activation in pre-B cells, strong activity throughout B cell ontogeny. To clarify its role, we generated mice with a clean deletion of the hs4 after replacement with a floxed neoR cassette. Surprisingly, and as for previous deletion of hs3a or hs1,2, deletion of hs4 did not affect either in vivo CSR or the secretion level of any Ig isotype. In vitro CSR and Ig secretion in response to LPS and cytokines was not affected either. The only noticeable effects of the hs4 deletion were a decrease in the number of B splenocytes and a decreased membrane IgM expression. In conclusion, while dispensable for CSR and Ig transcription in plasma cells, hs4 mostly appears to contribute to Ig transcription in resting B lymphocytes.

Recent evidence suggests that NK cells require priming to display full effector activity. In this study, we demonstrate that IL-18 contributed to this phenomenon. IL-18 signaling-deficient NK cells were found to be unable to secrete IFN-γ in response to ex vivo stimulation with IL-12. This was not due to a costimulatory role of IL-18, because blocking IL-18 signaling during the ex vivo stimulation with IL-12 did not alter IFN-γ production by wild-type NK cells. Rather, we demonstrate that IL-18 primes NK cells in vivo to produce IFN-γ upon subsequent stimulation with IL-12. Importantly, IL-12-induced IFN-γ transcription by NK cells was comparable in IL-18 signaling-deficient and -sufficient NK cells. This suggests that priming by IL-18 leads to an improved translation of IFN-γ mRNA. These results reveal a novel type of cooperation between IL-12 and IL-18 that requires the sequential action of these cytokines.

Host resistance to Trypanosoma cruzi infection depends on a type 1 response characterized by a strong production of IL-12 and IFN-γ. Amplifying this response through CD40 triggering results in control of parasitemia. Two newly synthesized molecules (

Bone marrow-derived mast cells (BMMC) have been used extensively as a mast cell model. BMMC, however, are immature cells that have no known physiological equivalent in tissues. They do not respond to IgG immune complexes. They may therefore not be appropriate for studying the physiopathology of IgE-induced allergies or IgG-induced tissue-specific inflammatory diseases which both depend on mature mast cells. Resident peritoneal mast cells are a minor population of differentiated cells that are not readily purified. They, however, can be expanded in culture to generate large numbers of homogeneous cells. We show here that these peritoneal cell-derived mast cells (PCMC) are mature serosal-type mouse mast cells which retain most morphological, phenotypic, and functional features of peritoneal mast cells. Like peritoneal mast cells, PCMC respond to IgG Abs. IgG immune complex-induced responses depended on FcγRIIIA and were negatively regulated by FcγRIIB. We found that a moderate FcγRIIB-dependent negative regulation, due not to a higher FcγRIIIA/FcγRIIB ratio, but to a relatively inefficient use of the lipid phosphatase SHIP1, determines this property of PCMC. PCMC also respond to IgE Abs. IgE-induced PCMC responses, however, differed quantitatively and qualitatively from BMMC responses. PCMC secreted no or much lower amounts of lipid mediators, chemokines, and cytokines, but they contained and released much higher amounts of preformed granular mediators. PCMC, but not BMMC, also contained and, upon degranulation, released molecules with a potent proteolytic activity. These properties make PCMC a useful new model for understanding the physiopathology of mast cells in IgE- and IgG-dependent tissue inflammation.

MHC-matched hemopoietic stem cell transplantation is commonly used for the treatment of some forms of leukemia. Conditioning regimens before transplant act to reduce the burden of leukemic cells and the graft-vs-leukemia (GvL) effect can eliminate residual disease. The GvL effect results largely from the recognition of minor histocompatibility Ags by donor T cells on recipient tissues. These Ags are generally widely expressed and also provoke graft-vs-host (GvH) disease. Manipulation of immunity to promote GvL while curtailing GvH would greatly improve clinical outcome. To develop strategies that may achieve this, the parameters which control immunity to minor histocompatibility Ags need to be defined. In this study, we have analyzed responses to the mouse HY minor histocompatibility Ag using hemopoietic cell and skin grafts as surrogate GvL and GvH targets, respectively. We show that natural regulation of CD8 T cell responses to HY operates at multiple levels. First, CD4 T cell help is required for primary CD8 responses directed at hemopoietic cells. However, although CD4 T cells of H2k mouse strains recognize HY, they provide ineffective help associated with a proportion of recipients developing tolerance. This was further investigated using TCR-transgenic mice which revealed H2k-restricted HY-specific CD4 T cells are highly susceptible to regulation by CD25+ regulatory T cells which expand in tolerant recipients. A second level of regulation, operating in the context of skin grafts, involves direct inhibition of CD8 T cell responses by CD94/NKG2 engagement of the nonclassical MHC class I molecule Qa1.

Lat Y136F knock-in mice harbor a point mutation in Tyr136 of the linker for activation of T cells and show accumulation of Th2 effector cells and IgG1 and IgE hypergammaglobulinemia. B cell activation is not a direct effect of the mutation on B cells since in the absence of T cells, mutant B cells do not show an activated phenotype. After adoptive transfer of linker for activation of T cell mutant T cells into wild-type, T cell-deficient recipients, recipient B cells become activated. We show in vivo and in vitro that the LatY136F mutation promotes T cell-dependent B cell activation leading to germinal center, memory, and plasma cell formation even in an MHC class II-independent manner. All the plasma and memory B cell populations found in physiological T cell-dependent B cell responses are found. Characterization of the abundant plasmablasts found in secondary lymphoid organs of LatY136F mice revealed the presence of a previously uncharacterized CD93-expressing subpopulation, whose presence was confirmed in wild-type mice after immunization. In LatY136F mice, B cell activation was polyclonal and not Ag-driven because the increase in serum IgG1 and IgE concentrations involved Abs and autoantibodies with different specificities equally. Although the noncomplement-fixing IgG1 and IgE are the only isotypes significantly increased in LatY136F serum, we observed early-onset systemic autoimmunity with nephritis showing IgE autoantibody deposits and severe proteinuria. These results show that Th2 cells developing in LatY136F mice can trigger polyclonal B cell activation and thereby lead to systemic autoimmune disease.

Long-term immunity to Leishmania may require the continued presence of parasites, but previous attempts to create attenuated parasites that persist without causing disease have had limited success. Since Leishmania major mutants that lack lipophosphoglycan and other secreted phosphoglycans, termed lpg2−, persist indefinitely in infected mice without inducing any disease, we tested their ability to provide protection to virulent L. major challenge. In response to leishmanial Ag stimulation, cells from lpg2−-infected mice produced minimal levels of IL-4 and IL-10, as well as very low levels of IFN-γ. Nevertheless, when BALB/c mice infected with lpg2− parasites were challenged with virulent L. major they were protected from disease. Thus, these findings report on attenuated parasites that may be used to induce long-term protection against leishmaniasis and indicate that the immunity induced can be maintained in the absence of a strong Th1 response.

We show that the lymphoid hyperplasia observed in IL-2Rα- and IL-2-deficient mice is due to the lack of a population of regulatory cells essential for CD4 T cell homeostasis. In chimeras reconstituted with bone marrow cells from IL-2Rα-deficient donors, restitution of a population of CD25+CD4+ T cells prevents the chaotic accumulation of lymphoid cells, and rescues the mice from autoimmune disease and death. The reintroduction of IL-2-producing cells in IL-2-deficient chimeras establishes a population of CD25+CD4+ T cells, and restores the peripheral lymphoid compartments to normal. The CD25+CD4+ T cells regulated selectively the number of naive CD4+ T cells transferred into T cell-deficient hosts. The CD25+CD4+/naive CD4 T cell ratio and the sequence of cell transfer determines the homeostatic plateau of CD4+ T cells. Overall, our findings demonstrate that IL-2Rα is an absolute requirement for the development of the regulatory CD25+CD4+ T cells that control peripheral CD4 T cell homeostasis, while IL-2 is required for establishing a sizeable population of these cells in the peripheral pools.

Dendritic cell-derived exosomes (DEX) are nanomeric vesicles harboring MHC/peptide complexes capable of promoting primary T cell responses and tumor rejection in the presence of adjuvants. In this study, we show that, in the absence of adjuvants, DEX mediate potent Ag-dependent antitumor effects against preestablished tumors in mice pretreated with immunopotentiating dosing of cyclophosphamide. Cyclophosphamide could 1) abolish the suppressive function of CD4+CD25+Foxp3+ regulatory T cells, 2) markedly enhance the magnitude of secondary but not primary CTL responses induced by DEX vaccines, 3) synergize with DEX in therapy but not prophylaxis tumor models. Therefore, therapeutic vaccines such as DEX aimed at boosting tumor-primed effector T cells could benefit procedures that minimize the effects of CD4+CD25+ regulatory T cells.