The role of antigen and IL-12 in sustaining Th1 memory cells in vivo: IL-12 is required to maintain memory/effector Th1 cells sufficient to mediate protection to an infectious parasite challenge

IL-12 plays a central role in both the induction and magnitude of a primary Th1 response. A critical question in designing vaccines for diseases requiring Th1 immunity such as Mycobacterium tuberculosis and Leishmania major is the requirements to sustain memory/effector Th1 cells in vivo. This report examines the role of IL-12 and antigen in sustaining Th1 responses sufficient for protective immunity to L. major after vaccination with LACK protein (LP) plus rIL-12 and LACK DNA. It shows that, after initial vaccination with LP plus rIL-12, supplemental boosting with either LP or rIL-12 is necessary but not sufficient to fully sustain long-term Th1 immunity. Moreover, endogenous IL-12 is also shown to be required for the induction, maintenance, and effector phase of the Th1 response after LACK DNA vaccination. Finally, IL-12 is required to sustain Th1 cells and control parasite growth in susceptible and resistant strains of mice during primary and secondary infection. Taken together, these data show that IL-12 is essential to sustain a sufficient number of memory/effector Th1 cells generated in vivo to mediate long-term protection to an intracellular pathogen.

Minimal epitopes expressed in a recombinant polyepitope protein are processed and presented to CD8+ cytotoxic T cells: implications for vaccine design.

The epitopes recognized by CD8+ cytotoxic T lymphocytes (CTL) are generated from cytosolic proteins by proteolytic processing. The nature of the influences exerted by the sequences flanking CTL epitopes on these processing events remains controversial. Here we show that each epitope within an artificial polyepitope protein containing nine minimal CD8+ CTL epitopes in sequence was processed and presented to appropriate CTL clones. Natural flanking sequences were thus not required to direct class I proteolytic processing. In addition, unnatural flanking sequences containing other CTL epitopes did not interfere with processing. The ability of every CTL epitope to be effectively processed from a protein containing only CTL epitopes is likely to find application in the construction of recombinant polyepitope CTL vaccines.

Fine mapping and functional analysis of the multiple sclerosis risk gene CD6

CD6 has recently been identified and validated as risk gene for multiple sclerosis (MS), based on the association of a single nucleotide polymorphism (SNP), rs17824933, located in intron 1. CD6 is a cell surface scavenger receptor involved in T-cell activation and proliferation, as well as in thymocyte differentiation. In this study, we performed a haptag SNP screen of the CD6 gene locus using a total of thirteen tagging SNPs, of which three were non-synonymous SNPs, and replicated the recently reported GWAS SNP rs650258 in a Spanish-Basque collection of 814 controls and 823 cases. Validation of the six most strongly associated SNPs was performed in an independent collection of 2265 MS patients and 2600 healthy controls. We identified association of haplotypes composed of two non-synonymous SNPs [rs11230563 (R225W) and rs2074225 (A257V)] in the 2nd SRCR domain with susceptibility to MS (Pmax(T) permutation=161024). The effect of these haplotypes on CD6 surface expression and cytokine secretion was also tested. The analysis showed significantly different CD6 expression patterns in the distinct cell subsets, i.e. ��� CD4+ na����ve cells, P = 0.0001; CD8+ na����ve cells, P,0.0001; CD4+ and CD8+ central memory cells, P = 0.01 and 0.05, respectively; and natural killer T (NKT) cells, P = 0.02; with the protective haplotype (RA) showing higher expression of CD6. However, no significant changes were observed in natural killer (NK) cells, effector memory and terminally differentiated effector memory T cells. Our findings reveal that this new MS-associated CD6 risk haplotype significantly modifies expression of CD6 on CD4+ and CD8+ T cells.

Participa����o das c��lulas Th1, Th2, Th17 e T regulat��rias na doen��a de Jorge Lobo: an��lise do perfil de express��o g��nica nas les��es

P��s-gradua����o em Doen��as Tropicais - FMB

Perfil de citocinas do padr��o Th1, Th2, Th17 e o estado redox de indiv��duos com leishmaniose visceral pr�� e p��s tratamento

Coordena����o de Aperfei��oamento de Pessoal de N��vel Superior (CAPES)

Cyclosporine A Drives a Th17���and Th2���Mediated Posttransplant Obliterative Airway Disease

Calcineurin-inhibitor refractory bronchiolitis obliterans (BO) represents the leading cause of late graft failure after lung transplantation. T helper (Th)2 and Th17 lymphocytes have been associated with BO development. Taking advantage of a fully allogeneic trachea transplantation model in mice, we addressed the pathogenicity of Th cells in obliterative airway disease (OAD) occurring in cyclosporine A (CsA)-treated recipients. We found that CsA prevented CD8+ T cell infiltration into the graft and downregulated the Th1 response but affected neither Th2 nor Th17 responses in vivo. In secondary mixed lymphocyte cultures, CsA dramatically decreased donor-specific IFN-�� production, enhanced IL-17 production and did not affect IL-13. As CD4+ depletion efficiently prevented OAD in CsA-treated recipients, we further explored the role of Th2 and Th17 immunity in vivo. Although IL-4 and IL-17 deficient untreated mice developed an OAD comparable to wild-type recipients, a single cytokine deficiency afforded significant protection in CsA-treated recipients. In conclusion, CsA treatment unbalances T helper alloreactivity and favors Th2 and Th17 as coexisting pathways mediating chronic rejection of heterotopic tracheal allografts.

TH2 cytokines from malignant cells suppress TH1 responses and enforce a global TH2 bias in leukemic cutaneous T-cell lymphoma

PURPOSE In leukemic cutaneous T-cell lymphoma (L-CTCL), malignant T cells accumulate in the blood and give rise to widespread skin inflammation. Patients have intense pruritus, increased immunoglobulin E (IgE), and decreased T-helper (TH)-1 responses, and most die from infection. Depleting malignant T cells while preserving normal immunity is a clinical challenge. L-CTCL has been variably described as a malignancy of regulatory, TH2 and TH17 cells. EXPERIMENTAL DESIGN We analyzed phenotype and cytokine production in malignant and benign L-CTCL T cells, characterized the effects of malignant T cells on healthy T cells, and studied the immunomodulatory effects of treatment modalities in patients with L-CTCL. RESULTS Twelve out of 12 patients with L-CTCL overproduced TH2 cytokines. Remaining benign T cells were also strongly TH2 biased, suggesting a global TH2 skewing of the T-cell repertoire. Culture of benign T cells away from the malignant clone reduced TH2 and enhanced TH1 responses, but separate culture had no effect on malignant T cells. Coculture of healthy T cells with L-CTCL T cells reduced IFN�� production and neutralizing antibodies to interleukin (IL)-4 and IL-13 restored TH1 responses. In patients, enhanced TH1 responses were observed following a variety of treatment modalities that reduced malignant T-cell burden. CONCLUSIONS A global TH2 bias exists in both benign and malignant T cells in L-CTCL and may underlie the infectious susceptibility of patients. TH2 cytokines from malignant cells strongly inhibited TH1 responses. Our results suggest that therapies that inhibit TH2 cytokine activity, by virtue of their ability to improve TH1 responses, may have the potential to enhance both anticancer and antipathogen responses.

Cutting edge: distinct glycolytic and lipid oxidative metabolic programs are essential for effector and regulatory CD4+ T cell subsets.

Stimulated CD4(+) T lymphocytes can differentiate into effector T cell (Teff) or inducible regulatory T cell (Treg) subsets with specific immunological roles. We show that Teff and Treg require distinct metabolic programs to support these functions. Th1, Th2, and Th17 cells expressed high surface levels of the glucose transporter Glut1 and were highly glycolytic. Treg, in contrast, expressed low levels of Glut1 and had high lipid oxidation rates. Consistent with glycolysis and lipid oxidation promoting Teff and Treg, respectively, Teff were selectively increased in Glut1 transgenic mice and reliant on glucose metabolism, whereas Treg had activated AMP-activated protein kinase and were dependent on lipid oxidation. Importantly, AMP-activated protein kinase stimulation was sufficient to decrease Glut1 and increase Treg generation in an asthma model. These data demonstrate that CD4(+) T cell subsets require distinct metabolic programs that can be manipulated in vivo to control Treg and Teff development in inflammatory diseases.

Optimal clearance of Sporothrix schenckii requires an intact Th17 response in a mouse model of systemic infection

Funda����o de Amparo �� Pesquisa do Estado de S��o Paulo (FAPESP)

Molekulare Charakterisierung der Il-9-Genexpression in T-Helfer-Typ-9-Zellen

CD4+ T-Zellen k��nnen in verschiedene T-Helferzellsubpopulationen differenzieren. Dabei h��ngt es von verschiedensten Milieubedingungen ab, welche Subpopulation sich auspr��gt, damit die CD4+ T-Zelle durch die Sekretion verschiedenster Zytokine ihre Funktion im Immunsystem wahrnehmen kann.rnBei der Th9-Subpopulation handelt es sich um einen IL-9-produzierenden Ph��notyp, welcher sich in der Anwesenheit von TGF-�� und IL-4 entwickelt39. Als treibender Transkriptionsfaktor f��r diese Subpopulation wurde das Protein IRF4 beschrieben45. Da dieser Transkriptionsfaktor auch f��r die Differenzierung weiterer Subpopulationen, wie Th2- und Th17-Zellen von Bedeutung ist30,121, stellte sich die Frage, welcher Interaktionspartner von IRF4 dar��ber entscheidet, welcher Subtyp sich entwickelt. Deshalb wurde in dieser Arbeit der Transkriptionsfaktor NFATc2 als m��glicher Interaktionspartner f��r IRF4 am murinen Il9 Promotor untersucht. Allerdings zeigten Reportergen��analysen, dass NFATc2 die IL-9-Produktion in Th9-Zellen inhibiert anstatt sie zu f��rdern. Th9-Zellen aus NFATc2-defizienten Tieren zeigen folglich im Vergleich zu wildtypischen Th9-Zellen sowohl nach Prim��r- als auch nach Restimulation eine verst��rkte IL-9-Produktion. Der Faktor NFATc2 kann somit als transkriptioneller Aktivator f��r die IL-9-Expression in Th9-Zellen ausgeschlossen werden. In vivo wurden diese Beobachtungen dadurch untermauert, dass NFATc2-defiziente Tiere im Rahmen des Asthma bronchiale zu einer verst��rkten pulmonalen Inflammation neigen und auch einen erh��hten Atemwegswiderstand nach Methacholin-Provokation aufweisen. Diese asthmatischen Symptome konnten durch Applikation eines neutralisierenden Antik��rpers f��r IL-9 wesentlich gemildert werden. In einem B16F10-Melanommodell konnten NFATc2-defiziente Tiere gegen��ber dem Wildtyp eine verbesserte anti-Tumorantwort auspr��gen. Nach Gabe eines IL-9-neutralisierenden Antik��rpers, wurde dieser Effekt wiederum gemildert.rnZusammenfassend l��sst sich sagen, dass IRF4 nicht mit NFATc2 am murinen Il9 Promotor interagiert, um die IL-9-Expression in Th9-Zellen zu f��rdern. Eine NFATc2-Defizienz resultiert sogar in einer gesteigerten IL-9-Produktion, womit ein inhibitorischer Einfluss von NFATc2 in Bezug auf die IL-9-Expression in Th9-Zellen nachgewiesen werden konnte.rn

Estrogen Receptor �� Signaling in T Lymphocytes Is Required for Estradiol-Mediated Inhibition of Th1 and Th17 Cell Differentiation and Protection against Experimental Autoimmune Encephalomyelitis

Estrogen treatment exerts a protective effect on experimental autoimmune encephalomyelitis (EAE) and is under clinical trial for multiple sclerosis therapy. Estrogens have been suspected to protect from CNS autoimmunity through their capacity to exert anti-inflammatory as well as neuroprotective effects. Despite the obvious impacts of estrogens on the pathophysiology of multiple sclerosis and EAE, the dominant cellular target that orchestrates the anti-inflammatory effect of 17��-estradiol (E2) in EAE is still ill defined. Using conditional estrogen receptor (ER) ��-deficient mice and bone marrow chimera experiments, we show that expression of ER�� is critical in hematopoietic cells but not in endothelial ones to mediate the E2 inhibitory effect on Th1 and Th17 cell priming, resulting in EAE protection. Furthermore, using newly created cell type-specific ER��-deficient mice, we demonstrate that ER�� is required in T lymphocytes, but neither in macrophages nor dendritic cells, for E2-mediated inhibition of Th1/Th17 cell differentiation and protection from EAE. Lastly, in absence of ER�� in host nonhematopoietic tissues, we further show that ER�� signaling in T cells is necessary and sufficient to mediate the inhibitory effect of E2 on EAE development. These data uncover T lymphocytes as a major and nonredundant cellular target responsible for the anti-inflammatory effects of E2 in Th17 cell-driven CNS autoimmunity.

Beta-arrestin-2 regulates the development of allergic asthma.

Asthma is a chronic inflammatory disorder of the airways that is coordinated by Th2 cells in both human asthmatics and animal models of allergic asthma. Migration of Th2 cells to the lung is key to their inflammatory function and is regulated in large part by chemokine receptors, members of the seven-membrane-spanning receptor family. It has been reported recently that T cells lacking beta-arrestin-2, a G protein-coupled receptor regulatory protein, demonstrate impaired migration in vitro. Here we show that allergen-sensitized mice having a targeted deletion of the beta-arrestin-2 gene do not accumulate T lymphocytes in their airways, nor do they demonstrate other physiological and inflammatory features characteristic of asthma. In contrast, the airway inflammatory response to LPS, an event not coordinated by Th2 cells, is fully functional in mice lacking beta-arrestin-2. beta-arrestin-2-deficient mice demonstrate OVA-specific IgE responses, but have defective macrophage-derived chemokine-mediated CD4+ T cell migration to the lung. This report provides the first evidence that beta-arrestin-2 is required for the manifestation of allergic asthma. Because beta-arrestin-2 regulates the development of allergic inflammation at a proximal step in the inflammatory cascade, novel therapies focused on this protein may prove useful in the treatment of asthma.

��-Arrestin-2 regulates the development of allergic asthma

Asthma is a chronic inflammatory disorder of the airways that is coordinated by Th2 cells in both human asthmatics and animal models of allergic asthma. Migration of Th2 cells to the lung is key to their inflammatory function and is regulated in large part by chemokine receptors, members of the seven-membrane-spanning receptor family. It has been reported recently that T cells lacking ��-arrestin-2, a G protein-coupled receptor regulatory protein, demonstrate impaired migration in vitro. Here we show that allergen-sensitized mice having a targeted deletion of the ��-arrestin-2 gene do not accumulate T lymphocytes in their airways, nor do they demonstrate other physiological and inflammatory features characteristic of asthma. In contrast, the airway inflammatory response to LPS, an event not coordinated by Th2 cells, is fully functional in mice lacking ��-arrestin-2. ��-arrestin-2-deficient mice demonstrate OVA-specific IgE responses, but have defective macrophage-derived chemokine-mediated CD4+ T cell migration to the lung. This report provides the first evidence that ��-arrestin-2 is required for the manifestation of allergic asthma. Because ��-arrestin-2 regulates the development of allergic inflammation at a proximal step in the inflammatory cascade, novel therapies focused on this protein may prove useful in the treatment of asthma.

Innate immunogenicity and in vitro protective potential of Schistosoma mansoni lung schistosomula excretory-secretory candidate vaccine antigens

Excretory secretory products (ESP) of Schistosoma mansoni developing larvae are ideal potential vaccines as such molecules may readily induce host primary immune responses, and local memory immune response effectors that would target, surround, and pursue the larvae while negotiating the lung blood capillaries. We herein characterized the cytokines response ESP, e.g., SG3PDH, 14-3-3-like protein, TPX, and calpain induce in the natural context of infection, and defined the global cytokine profile conducive to effective schistosome larvae killing. Accordingly, spleen cells (SC) taken from naive, and 7-, or 9-day S. mansoni-infected mice were stimulated in vitro with the selected ESP, in a recombinant or multiple antigen peptide (MAP) form, and examined for production of T helper type (Th) 1, Th2, and Th17 cytokines, and the ability to mediate in vitro attrition of lung-stage schistosomula. The study indicated that larval ESP principally elicit Th1 and Th17 type cytokines. Recombinant SG3PDH was the only test ESP to additionally activate SC from S. mansoni-infected BALB/c mice to release higher IL-4 levels than unstimulated SC and mediate significant (P

��tude de protection conf��r��e par vaccination ADN dans un mod��le murin d���h��patite auto-immune

La vaccination ADN �� l���aide de plasmides codant pour des autoantig��nes s���est av��r��e efficace dans la protection contre plusieurs maladies auto-immunes. Le but de ce m��moire ��tait dans un premier temps d�����tablir si un protocole de vaccination ADN compos�� de 3 injections de pCMV-CTLA-4-NP et de pVR-IL-12 �� deux semaines d���intervalle avait un effet protecteur contre le d��veloppement d���une h��patite auto-immune chez la souris TTR-NP, un mod��le murin transg��nique de la maladie et pr��c��demment d��velopp�� au laboratoire. Dans un deuxi��me temps, le but ��tait d�����lucider, le cas ��ch��ant, les m��canismes sous-tendant la protection conf��r��e par la vaccination ADN. Les hypoth��ses initiales ��taient qu���une protection allait effectivement ��tre conf��r��e par la vaccination ADN et que celle-ci pouvait ��tre attribuable �� une d��viation de la r��ponse typiquement Th1 de la maladie vers une r��ponse Th2, �� un ��puisement des cellules immunitaires et/ou �� l���activation et �� l���induction de prolif��ration de cellules r��gulatrices. Les r��sultats montrent que la vaccination ADN induit une protection transitoire contre le d��veloppement d���infiltrations lymphocytaires au foie. Cette protection se ferait via un ��puisement des cellules CD4+, CD8+ et CD19+ se retrouvant �� la rate et exprimant PD 1 dans une plus forte proportion �� 3 mois, et ne serait m��di��e ni par les lymphocytes T r��gulateurs CD4+CD25+FoxP3+, ni par les cellules CD8+FoxP3+. Une d��viation de la r��ponse Th1 vers une r��ponse Th2 demeure une explication suppl��mentaire plausible �� la protection conf��r��e mais n��cessiterait une caract��risation en situation plus physiologique avant de pouvoir inf��rer sur son implication r��elle. La vaccination ADN n���influe ni sur la pr��sence d���autoanticorps, ni sur les niveaux d���alanine aminotransf��rase, deux marqueurs de la maladie.