Interleukin-17 is a critical mediator of vaccine-induced reduction of Helicobacter infection in the mouse model.

BACKGROUND & AIMS: Despite the proven ability of immunization to reduce Helicobacter infection in mouse models, the precise mechanism of protection has remained elusive. This study explores the possibility that interleukin (IL)-17 plays a role in the reduction of Helicobacter infection following vaccination of wild-type animals or in spontaneous reduction of bacterial infection in IL-10-deficient mice. METHODS: In mice, reducing Helicobacter infection, the levels and source of IL-17 were determined and the role of IL-17 in reduction of Helicobacter infection was probed by neutralizing antibodies. RESULTS: Gastric IL-17 levels were strongly increased in mice mucosally immunized with urease plus cholera toxin and challenged with Helicobacter felis as compared with controls (654 +/- 455 and 34 +/- 84 relative units for IL-17 messenger RNA expression [P < .01] and 6.9 +/- 8.4 and 0.02 +/- 0.04 pg for IL-17 protein concentration [P < .01], respectively). Flow cytometry analysis showed that a peak of CD4(+)IL-17(+) T cells infiltrating the gastric mucosa occurred in immunized mice in contrast to control mice (4.7% +/- 0.3% and 1.4% +/- 0.3% [P < .01], respectively). Gastric mucosa-infiltrating CD4(+)IL-17(+) T cells were also observed in IL-10-deficient mice that spontaneously reduced H felis infection (4.3% +/- 2.3% and 2% +/- 0.6% [P < .01], for infected and noninfected IL-10-deficient mice, respectively). In wild-type immunized mice, intraperitoneal injection of anti-IL-17 antibodies significantly inhibited inflammation and the reduction of Helicobacter infection in comparison with control antibodies (1 of 12 mice vs 9 of 12 mice reduced Helicobacter infection [P < .01], respectively). CONCLUSIONS: IL-17 plays a critical role in the immunization-induced reduction of Helicobacter infection from the gastric mucosa.

The IL-4 rapidly produced in BALB/c mice after infection with Leishmania major down-regulates IL-12 receptor beta 2-chain expression on CD4+ T cells resulting in a state of unresponsiveness to IL-12.

Within 1 day of infection with Leishmania major, susceptible BALB/c mice produce a burst of IL-4 in their draining lymph nodes, resulting in a state of unresponsiveness to IL-12 in parasite-specific CD4+ T cells within 48 h. In this report we examined the molecular mechanism underlying this IL-12 unresponsiveness. Extinction of IL-12 signaling in BALB/c mice is due to a rapid down-regulation of IL-12R beta2-chain mRNA expression in CD4+ T cells. In contrast, IL-12R beta2-chain mRNA expression was maintained on CD4+ T cells from resistant C57BL/6 mice. The down-regulation of the IL-12R beta2-chain mRNA expression in BALB/c CD4+ T cells is a consequence of the early IL-4 production. In this murine model of infection, a strict correlation is shown in vivo between expression of the IL-12R beta2-chain in CD4+ T cells and the development of a Th1 response and down-regulation of the mRNA beta2-chain expression and the maturation of a Th2 response. Treatment of BALB/c mice with IFN-gamma, even when IL-4 has been produced for 48 h, resulted in maintenance of IL-12R beta2-chain mRNA expression and IL-12 responsiveness. The data presented here support the hypothesis that the genetically determined susceptibility of BALB/c mice to infection with L. major is primarily based on an up-regulation of IL-4 production, which secondarily induces extinction of IL-12 signaling.

CD8(+) T cells specific for tumor antigens can be rendered dysfunctional by the tumor microenvironment through upregulation of the inhibitory receptors BTLA and PD-1.

Cytotoxic T cells that are present in tumors and capable of recognizing tumor epitopes are nevertheless generally impotent in eliciting tumor rejection. Thus, identifying the immune escape mechanisms responsible for inducing tumor-specific CD8(+) T-cell dysfunction may reveal effective strategies for immune therapy. The inhibitory receptors PD-1 and Tim-3 are known to negatively regulate CD8(+) T-cell responses directed against the well-characterized tumor antigen NY-ESO-1. Here, we report that the upregulation of the inhibitory molecule BTLA also plays a critical role in restricting NY-ESO-1-specific CD8(+) T-cell expansion and function in melanoma. BTLA-expressing PD-1(+)Tim-3(-) CD8(+) T cells represented the largest subset of NY-ESO-1-specific CD8(+) T cells in patients with melanoma. These cells were partially dysfunctional, producing less IFN-γ than BTLA(-) T cells but more IFN-γ, TNF, and interleukin-2 than the highly dysfunctional subset expressing all three receptors. Expression of BTLA did not increase with higher T-cell dysfunction or upon cognate antigen stimulation, as it does with PD-1, suggesting that BTLA upregulation occurs independently of functional exhaustion driven by high antigen load. Added with PD-1 and Tim-3 blockades, BTLA blockade enhanced the expansion, proliferation, and cytokine production of NY-ESO-1-specific CD8(+) T cells. Collectively, our findings indicate that targeting BTLA along with the PD-1 and Tim-3 pathways is critical to reverse an important mechanism of immune escape in patients with advanced melanoma.

Gene transfer of a soluble IL-1 type 2 receptor-Ig fusion protein improves cardiac allograft survival in rats.

OBJECTIVE: Interleukin-1 (IL-1) mediates ischemia-reperfusion injury and graft inflammation after heart transplantation. IL-1 affects target cells through two distinct types of transmembrane receptors, type-1 receptor (IL-1R1), which transduces the signal, and the non-signaling type-2 receptor (IL-1R2), which acts as a ligand sink that subtracts IL-1beta from IL-1R1. We analyzed the efficacy of adenovirus (Ad)-mediated gene transfer of a soluble IL-1R2-Ig fusion protein in delaying cardiac allograft rejection and the mechanisms underlying the protective effect. METHODS: IL-1 inhibition by IL-1R2-Ig was tested using an in vitro functional assay whereby endothelial cells preincubated with AdIL-1R2-Ig or control virus were stimulated with recombinant IL-1beta or tumor necrosis factor-alpha (TNF-alpha), and urokinase-type plasminogen activator (u-PA) induction was measured by zymography. AdIL-1R2-Ig was delivered to F344 rat donor hearts ex vivo, which were placed in the abdominal position in LEW hosts. Intragraft inflammatory cell infiltrates and proinflammatory cytokine expression were analyzed by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. RESULTS: IL-1R2-Ig specifically inhibited IL-1beta-induced u-PA responses in vitro. IL-1R2-Ig gene transfer reduced intragraft monocytes/macrophages and CD4(+) cell infiltrates (p<0.05), TNF-alpha and transforming growth factor-beta (TGF-beta) expression (p<0.05), and prolonged graft survival (15.6+/-5.7 vs 10.3+/-2.5 days with control vector and 10.1+/-2.1 days with buffer alone; p<0.01). AdIL-1R2-Ig combined with a subtherapeutic regimen of cyclosporin A (CsA) was superior to CsA alone (19.4+/-3.0 vs 15.9+/-1.8 days; p<0.05). CONCLUSIONS: Soluble IL-1 type-2 receptor gene transfer attenuates cardiac allograft rejection in a rat model. IL-1 inhibition may be useful as an adjuvant therapy in heart transplantation.

Interleukin-22 is increased in multiple sclerosis patients and targets astrocytes.

BACKGROUND: Increasing evidences link T helper 17 (Th17) cells with multiple sclerosis (MS). In this context, interleukin-22 (IL-22), a Th17-linked cytokine, has been implicated in blood brain barrier breakdown and lymphocyte infiltration. Furthermore, polymorphism between MS patients and controls has been recently described in the gene coding for IL-22 binding protein (IL-22BP). Here, we aimed to better characterize IL-22 in the context of MS. METHODS: IL-22 and IL-22BP expressions were assessed by ELISA and qPCR in the following compartments of MS patients and control subjects: (1) the serum, (2) the cerebrospinal fluid, and (3) immune cells of peripheral blood. Identification of the IL-22 receptor subunit, IL-22R1, was performed by immunohistochemistry and immunofluorescence in human brain tissues and human primary astrocytes. The role of IL-22 on human primary astrocytes was evaluated using 7-AAD and annexin V, markers of cell viability and apoptosis, respectively. RESULTS: In a cohort of 141 MS patients and healthy control (HC) subjects, we found that serum levels of IL-22 were significantly higher in relapsing MS patients than in HC but also remitting and progressive MS patients. Monocytes and monocyte-derived dendritic cells contained an enhanced expression of mRNA coding for IL-22BP as compared to HC. Using immunohistochemistry and confocal microscopy, we found that IL-22 and its receptor were detected on astrocytes of brain tissues from both control subjects and MS patients, although in the latter, the expression was higher around blood vessels and in MS plaques. Cytometry-based functional assays revealed that addition of IL-22 improved the survival of human primary astrocytes. Furthermore, tumor necrosis factor α-treated astrocytes had a better long-term survival capacity upon IL-22 co-treatment. This protective effect of IL-22 seemed to be conferred, at least partially, by a decreased apoptosis. CONCLUSIONS: We show that (1) there is a dysregulation in the expression of IL-22 and its antagonist, IL-22BP, in MS patients, (2) IL-22 targets specifically astrocytes in the human brain, and (3) this cytokine confers an increased survival of the latter cells.

Mammalian Target of Rapamycin Complex 2 Controls CD8 T Cell Memory Differentiation in a Foxo1-Dependent Manner.

Upon infection, antigen-specific naive CD8 T cells are activated and differentiate into short-lived effector cells (SLECs) and memory precursor cells (MPECs). The underlying signaling pathways remain largely unresolved. We show that Rictor, the core component of mammalian target of rapamycin complex 2 (mTORC2), regulates SLEC and MPEC commitment. Rictor deficiency favors memory formation and increases IL-2 secretion capacity without dampening effector functions. Moreover, mTORC2-deficient memory T cells mount more potent recall responses. Enhanced memory formation in the absence of mTORC2 was associated with Eomes and Tcf-1 upregulation, repression of T-bet, enhanced mitochondrial spare respiratory capacity, and fatty acid oxidation. This transcriptional and metabolic reprogramming is mainly driven by nuclear stabilization of Foxo1. Silencing of Foxo1 reversed the increased MPEC differentiation and IL-2 production and led to an impaired recall response of Rictor KO memory T cells. Therefore, mTORC2 is a critical regulator of CD8 T cell differentiation and may be an important target for immunotherapy interventions.

NALP3 is not necessary for early protection against experimental tuberculosis.

In vitro, Toll-like receptors (TLR)2, 4 and 9 as well as NOD-like receptor 2 critically determine macrophage responses to Mycobacterium tuberculosis (Mtb) infection. However, in low-dose experimental murine tuberculosis, single or multiple deficiencies in TLRs 2, 4, 9 or NOD2 have little, if any, impact on early mycobacterial growth containment, granuloma formation and survival. Here, we analyzed the relevance of NALP3, one component of the danger-signaling inflammasome, for (i) Mtb-induced cytokine secretion in vitro and in vivo, (ii) restriction of Mtb replication in infected organs and (iii) granuloma formation. In the absence of functional NALP3, there was no IL-1beta and IL-18 production in Mtb-infected dendritic cells and macrophages in vitro, whereas secretion of IL-1alpha, IL-12p40 and TNF remained unaffected. After three weeks of infection, NALP3-deficient as well as IL-18-deficient mice were as capable as wildtype mice of restricting Mtb loads at a plateau level within well-differentiated granulomas. In conclusion, despite its involvement in cytokine processing, NALP3 is not essential for induction of protective immunity to Mtb.

IL-2- and CD25-dependent immunoregulatory mechanisms in the homeostasis of T-cell subsets.

IL-2 plays a pivotal role in regulating the adaptive immune system by controlling the survival and proliferation of regulatory T (Treg) cells, which are required for the maintenance of immune tolerance. Moreover, IL-2 is implicated in the differentiation and homeostasis of effector T-cell subsets, including T(H)1, T(H)2, T(H)17, and memory CD8+ T cells. The IL-2 receptor is composed of 3 distinct subunits, namely the alpha (CD25), beta (CD122), and gamma (gammac) chains. Of crucial importance for the delivery of IL-2 signals to Treg cells is the expression of CD25, which, along with CD122 and gammac, confers high affinity binding to IL-2. Notably, recent findings suggest a novel role for CD25, whereby CD25 molecules on Treg cells and possibly other cells are capable of influencing T-cell homeostasis by means of IL-2 deprivation. This review explores these findings and integrates them into our current understanding of T-cell homeostasis.

The Interleukin-1 receptor antagonist is a direct target gene of PPARalpha in liver.

BACKGROUND/AIMS: The Peroxisome Proliferator-Activated Receptor (PPAR) alpha belongs to the superfamily of Nuclear Receptors and plays an important role in numerous cellular processes, including lipid metabolism. It is known that PPARalpha also has an anti-inflammatory effect, which is mainly achieved by down-regulating pro-inflammatory genes. The objective of this study was to further characterize the role of PPARalpha in inflammatory gene regulation in liver. RESULTS: According to Affymetrix micro-array analysis, the expression of various inflammatory genes in liver was decreased by treatment of mice with the synthetic PPARalpha agonist Wy14643 in a PPARalpha-dependent manner. In contrast, expression of Interleukin-1 receptor antagonist (IL-1ra), which was acutely stimulated by LPS treatment, was induced by PPARalpha. Up-regulation of IL-1ra by LPS was lower in PPARalpha -/- mice compared to Wt mice. Transactivation and chromatin immunoprecipitation studies identified IL-1ra as a direct positive target gene of PPARalpha with a functional PPRE present in the promoter. Up-regulation of IL-1ra by PPARalpha was conserved in human HepG2 hepatoma cells and the human monocyte/macrophage THP-1 cell line. CONCLUSIONS: In addition to down-regulating expression of pro-inflammatory genes, PPARalpha suppresses the inflammatory response by direct up-regulation of genes with anti-inflammatory properties.

LACK-reactive CD4+ T cells require autocrine IL-2 to mediate susceptibility to Leishmania major.

Mice from most inbred strains are resistant to infection with Leishmania major whereas mice from BALB strains are highly susceptible. Resistance and susceptibility result from the development of Th1 or Th2 cells, respectively. In this report, we document an IL-2 mRNA burst, preceding the reported early IL-4 response, in draining lymph nodes of susceptible mice infected with L. major. Neutralization of IL-2 during the first days of infection redirected Th1 cell maturation and resistance to L. major, through interference with the rapid IL-4 transcription in Leishmania homolog of mammalian RACK1 (LACK)-reactive CD4(+) cells. A burst of IL-2 transcripts also occurred in infected C57BL/6 mice that do not mount an early IL-4 response. However, although the LACK protein induced IL-2 transcripts in susceptible mice, it failed to trigger this response in resistant C57BL/6 mice. Reconstitution experiments using C.B.-17 SCID mice and LACK-reactive CD4(+) T cells from IL-2(-/-) BALB/c mice showed that triggering of the early IL-4 response required autocrine IL-2. Thus, in C57BL/6 mice, the inability of LACK-reactive CD4(+) T cells to express early IL-4 mRNA transcription, important for disease progression, appears due to an incapacity of these cells to produce IL-2.

Proliferation capacity and cytotoxic activity are mediated by functionally and phenotypically distinct virus-specific CD8 T cells defined by interleukin-7R{alpha} (CD127) and perforin expression.

Cytotoxicity and proliferation capacity are key functions of antiviral CD8 T cells. In the present study, we investigated a series of markers to define these functions in virus-specific CD8 T cells. We provide evidence that there is a lack of coexpression of perforin and CD127 in human CD8 T cells. CD127 expression on virus-specific CD8 T cells correlated positively with proliferation capacity and negatively with perforin expression and cytotoxicity. Influenza virus-, cytomegalovirus-, and Epstein-Barr virus/human immunodeficiency virus type 1-specific CD8 T cells were predominantly composed of CD127(+) perforin(-)/CD127(-) perforin(+), and CD127(-)/perforin(-) CD8 T cells, respectively. CD127(-)/perforin(-) and CD127(-)/perforin(+) cells expressed significantly more PD-1 and CD57, respectively. Consistently, intracellular cytokine (gamma interferon, tumor necrosis factor alpha, and interleukin-2 [IL-2]) responses combined to perforin detection confirmed that virus-specific CD8 T cells were mostly composed of either perforin(+)/IL-2(-) or perforin(-)/IL-2(+) cells. In addition, perforin expression and IL-2 secretion were negatively correlated in virus-specific CD8 T cells (P &lt; 0.01). As previously shown for perforin, changes in antigen exposure modulated also CD127 expression. Based on the above results, proliferating (CD127(+)/IL-2-secreting) and cytotoxic (perforin(+)) CD8 T cells were contained within phenotypically distinct T-cell populations at different stages of activation or differentiation and showed different levels of exhaustion and senescence. Furthermore, the composition of proliferating and cytotoxic CD8 T cells for a given antiviral CD8 T-cell population appeared to be influenced by antigen exposure. These results advance our understanding of the relationship between cytotoxicity, proliferation capacity, the levels of senescence and exhaustion, and antigen exposure of antiviral memory CD8 T cells.

CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation.

CARMA1 is a lymphocyte-specific member of the membrane-associated guanylate kinase (MAGUK) family of scaffolding proteins, which coordinate signaling pathways emanating from the plasma membrane. CARMA1 interacts with Bcl10 via its caspase-recruitment domain (CARD). Here we investigated the role of CARMA1 in T cell activation and found that T cell receptor (TCR) stimulation induced a physical association of CARMA1 with the TCR and Bcl10. We found that CARMA1 was constitutively associated with lipid rafts, whereas cytoplasmic Bcl10 translocated into lipid rafts upon TCR engagement. A CARMA1 mutant, defective for Bcl10 binding, had a dominant-negative (DN) effect on TCR-induced NF-kappa B activation and IL-2 production and on the c-Jun NH(2)-terminal kinase (Jnk) pathway when the TCR was coengaged with CD28. Together, our data show that CARMA1 is a critical lipid raft-associated regulator of TCR-induced NF-kappa B activation and CD28 costimulation-dependent Jnk activation.

Oligoclonal expansions in the CD8(+)CD28(-) T cells largely explain the shorter telomeres detected in this subset: analysis by flow FISH.

We have previously reported that CD8(+)CD28(-) T cells have relatively shorter telomeres compared with CD8(+)CD28(+) T cells. Oligoclonal expansion is a common feature of CD8(+) T cells in human peripheral blood, and these expansions predominantly occur in the CD57(+)/CD28(-) population. We studied the telomere length in subsets of CD8(+) T cells using quantitative fluorescence in situ hybridization and flow cytometry (flow FISH). Our results confirm that CD8(+)CD28(-) T cells have shorter telomeres as compared with their CD28(+) counterpart cells. In addition, the oligoclonally expanded cells within the CD8(+)CD28(-) T cell subset generally have even shorter telomeres than the CD28(-) subset as a whole. We conclude that the presence of clonal expansions in the CD8(+)CD28(-) T cell population largely explain the shorter telomeres in this subset. These clonally expanded CD8(+)CD28(-) T cells generally have characteristics of terminally differentiated effector cells. Nevertheless, there is considerable individual variation in the degree of telomere shortening in these cells, which may reflect host genetic factors as well as the type and timing of the antigenic exposure.

Effector function of human tumor-specific CD8 T cells in melanoma lesions: a state of local functional tolerance.

Although tumor-specific CD8 T-cell responses often develop in cancer patients, they rarely result in tumor eradication. We aimed at studying directly the functional efficacy of tumor-specific CD8 T cells at the site of immune attack. Tumor lesions in lymphoid and nonlymphoid tissues (metastatic lymph nodes and soft tissue/visceral metastases, respectively) were collected from stage III/IV melanoma patients and investigated for the presence and function of CD8 T cells specific for the tumor differentiation antigen Melan-A/MART-1. Comparative analysis was conducted with peripheral blood T cells. We provide evidence that in vivo-priming selects, within the available naive Melan-A/MART-1-specific CD8 T-cell repertoire, cells with high T-cell receptor avidity that can efficiently kill melanoma cells in vitro. In vivo, primed Melan-A/MART-1-specific CD8 T cells accumulate at high frequency in both lymphoid and nonlymphoid tumor lesions. Unexpectedly, however, whereas primed Melan-A/MART-1-specific CD8 T cells that circulate in the blood display robust inflammatory and cytotoxic functions, those that reside in tumor lesions (particularly in metastatic lymph nodes) are functionally tolerant. We show that both the lymph node and the tumor environments blunt T-cell effector functions and offer a rationale for the failure of tumor-specific responses to effectively counter tumor progression.

Cutting edge: IL-7 regulates the peripheral pool of adult ROR gamma+ lymphoid tissue inducer cells.

During fetal life, CD4(+)CD3(-) lymphoid tissue inducer (LTi) cells are required for lymph node and Peyer's patch development in mice. In adult animals, CD4(+)CD3(-) cells are found in low numbers in lymphoid organs. Whether adult CD4(+)CD3(-) cells are LTi cells and are generated and maintained through cytokine signals has not been directly addressed. In this study we show that adult CD4(+)CD3(-) cells adoptively transferred into neonatal CXCR5(-/-) mice induced the formation of intestinal lymphoid tissues, demonstrating for the first time their bona fide LTi function. Increasing IL-7 availability in wild-type mice either by IL-7 transgene expression or treatment with IL-7/anti-IL-7 complexes increased adult LTi cell numbers through de novo generation from bone marrow cells and increased the survival and proliferation of LTi cells. Our observations demonstrate that adult CD4(+)lineage(-) cells are LTi cells and that the availability of IL-7 determines the size of the adult LTi cell pool.