Individuals who are immunocompromised, including AIDS patients with few CD4+ T cells, are at increased risk for opportunistic fungal infections. The incidence of such infections is increasing worldwide, meaning that the need for antifungal vaccines is increasing. Although CD4+ T cells play a dominant role in resistance to many pathogenic fungal infections, we have previously shown that vaccination can induce protective antifungal CD8+ T cell immunity in the absence of CD4+ T cells. However, it has not been determined whether vaccine-induced antifungal CD8+ T cell memory can be maintained in the absence of CD4+ T cell help. Here, we have shown in a mouse model of vaccination against blastomycosis that antifungal memory CD8+ T cells are maintained in the absence of CD4+ T cells without loss of numbers or function for at least 6 months and that the cells protect against infection. Using a system that enabled us to induce and track antigen-specific, antifungal CD8+ T cells, we found that such cells were maintained for at least 5 months upon transfer into naive mice lacking both CD4+ T cells and persistent fungal antigen. Additionally, fungal vaccination induced a profile of transcription factors functionally linked with persistent memory in CD8+ T cells. Thus, unlike bacteria and viruses, fungi elicit long-term CD8+ T cell memory that is maintained without CD4+ T cell help or persistent antigen. This has implications for the development of novel antifungal vaccine strategies effective in immunocompromised patients.
Som G. Nanjappa, Erika Heninger, Marcel Wüthrich, Thomas Sullivan, Bruce Klein
cAMP, the intracellular signaling molecule produced in response to GPCR signaling, has long been recognized as an immunosuppressive agent that inhibits T cell receptor activation and T cell function. However, recent studies show that cAMP also promotes T cell–mediated immunity. Central to cAMP production downstream of GPCR activation is the trimeric G protein Gs. In order to reconcile the reports of divergent effects of cAMP in T cells and to define the direct effect of cAMP in T cells, we engineered mice in which the stimulatory Gα subunit of Gs (Gαs) could be deleted in T cells using CD4-Cre (GnasΔCD4). GnasΔCD4 CD4+ T cells had reduced cAMP accumulation and Ca2+ influx. In vitro and in vivo, GnasΔCD4 CD4+ T cells displayed impaired differentiation to specific Th subsets: Th17 and Th1 cells were reduced or absent, but Th2 and regulatory T cells were unaffected. Furthermore, GnasΔCD4 CD4+ T cells failed to provoke colitis in an adoptive transfer model, indicating reduced inflammatory function. Restoration of cAMP levels rescued the impaired phenotype of GnasΔCD4 CD4+ T cells, reinstated the PKA-dependent influx of Ca2+, and enhanced the ability of these cells to induce colitis. Our findings thus define an important role for cAMP in the differentiation of Th subsets and their subsequent inflammatory responses, and provide evidence that altering cAMP levels in CD4+ T cells could provide an immunomodulatory approach targeting specific Th subsets.
Xiangli Li, Fiona Murray, Naoki Koide, Jonathan Goldstone, Sara M. Dann, Jianzhong Chen, Samuel Bertin, Guo Fu, Lee S. Weinstein, Min Chen, Maripat Corr, Lars Eckmann, Paul A. Insel, Eyal Raz
Neutrophils are essential for maintaining innate immune surveillance under normal conditions, but also represent a major contributor to tissue damage during inflammation. Neutrophil homeostasis is therefore tightly regulated. Cxcr2 plays a critical role in neutrophil homeostasis, as Cxcr2–/– mice demonstrate mild neutrophilia and severe neutrophil hyperplasia in the bone marrow. The mechanisms underlying these phenotypes, however, are unclear. We report here that Cxcr2 on murine neutrophils inhibits the IL-17A/G-CSF axis that regulates neutrophil homeostasis. Furthermore, enterocyte-derived Cxcl5 in the gut regulates IL-17/G-CSF levels and contributes to Cxcr2-dependent neutrophil homeostasis. Conversely, G-CSF was required for Cxcl5-dependent regulation of neutrophil homeostasis, and inhibition of IL-17A reduced plasma G-CSF concentrations and marrow neutrophil numbers in both Cxcl5–/– and Cxcr2–/– mice. Cxcr2–/– mice constitutively expressed IL-17A and showed increased numbers of IL-17A–producing cells in the lung, terminal ileum, and spleen. Most IL-17–producing splenocytes were responsive to IL-1β plus IL-23 in vitro. Depletion of commensal microbes by antibiotic treatment in Cxcr2–/– mice markedly decreased IL-17A and G-CSF expression, neutrophilia, and marrow myeloid hyperplasia. These data suggest a critical role for Cxcr2, Cxcl5, and commensal bacteria in regulation of the IL-17/G-CSF axis and neutrophil homeostasis at mucosal sites and have implications for the development of treatments for pathologies resulting from either excessive or ineffective neutrophil responses.
Junjie Mei, Yuhong Liu, Ning Dai, Christian Hoffmann, Kristin M. Hudock, Peggy Zhang, Susan H. Guttentag, Jay K. Kolls, Paula M. Oliver, Frederic D. Bushman, G. Scott Worthen
Appropriate localization and migration of T cells is a prerequisite for antitumor immune surveillance. Studies using fixed tumor samples from human patients have shown that T cells accumulate more efficiently in the stroma than in tumor islets, but the mechanisms by which this occurs are unknown. By combining immunostaining and real-time imaging in viable slices of human lung tumors, we revealed that the density and the orientation of the stromal extracellular matrix likely play key roles in controlling the migration of T cells. Active T cell motility, dependent on chemokines but not on β1 or β2 integrins, was observed in loose fibronectin and collagen regions, whereas T cells migrated poorly in dense matrix areas. Aligned fibers in perivascular regions and around tumor epithelial cell regions dictated the migratory trajectory of T cells and restricted them from entering tumor islets. Consistently, matrix reduction with collagenase increased the ability of T cells to contact cancer cells. Thus, the stromal extracellular matrix influences antitumor immunity by controlling the positioning and migration of T cells. Understanding the mechanisms by which this collagen network is generated has the potential to aid in the development of new therapeutics.
Hélène Salmon, Katarzyna Franciszkiewicz, Diane Damotte, Marie-Caroline Dieu-Nosjean, Pierre Validire, Alain Trautmann, Fathia Mami-Chouaib, Emmanuel Donnadieu
Suppression of immune responses is necessary to limit damage to host tissue during inflammation, but it can be detrimental in specific immune responses, such as sepsis and antitumor immunity. Recently, immature myeloid cells have been implicated in the suppression of immune responses in mouse models of cancer, infectious disease, bone marrow transplantation, and autoimmune disease. Here, we report the identification of a subset of mature human neutrophils (CD11cbright/CD62Ldim/CD11bbright/CD16bright) as what we believe to be a unique circulating population of myeloid cells, capable of suppressing human T cell proliferation. These cells were observed in humans in vivo during acute systemic inflammation induced by endotoxin challenge or by severe injury. Local release of hydrogen peroxide from the neutrophils into the immunological synapse between the neutrophils and T cells mediated the suppression of T cell proliferation and required neutrophil expression of the integrin Mac-1 (αMβ2). Our data demonstrate that suppression of T cell function can be accomplished by a subset of human neutrophils that can be systemically induced in response to acute inflammation. Identification of the pivotal role of neutrophil Mac-1 and ROS in this process provides a potential target for modulating immune responses in humans.
Janesh Pillay, Vera M. Kamp, Els van Hoffen, Tjaakje Visser, Tamar Tak, Jan-Willem Lammers, Laurien H. Ulfman, Luke P. Leenen, Peter Pickkers, Leo Koenderman
The most abundant immune cell type is the neutrophil, a key first responder after pathogen invasion. Neutrophil numbers in the periphery are tightly regulated to prevent opportunistic infections and aberrant inflammation. In healthy individuals, more than 1 × 109 neutrophils per kilogram body weight are released from the bone marrow every 24 hours. To maintain homeostatic levels, an equivalent number of senescent cells must be cleared from circulation. Recent studies indicate that clearance of senescent neutrophils by resident tissue macrophages and DCs helps to set homeostatic levels of neutrophils via effects on the IL-23/IL-17/G-CSF cytokine axis, which stimulates neutrophil production in the bone marrow. However, the molecular events in phagocytes underlying this feedback loop have remained indeterminate. Liver X receptors (LXRs) are members of the nuclear receptor superfamily that regulate both lipid metabolic and inflammatory gene expression. Here, we demonstrate that LXRs contribute to the control of neutrophil homeostasis. Using gain- and loss-of-function models, we found that LXR signaling regulated the efficient clearance of senescent neutrophils by peripheral tissue APCs in a Mer-dependent manner. Furthermore, activation of LXR by engulfed neutrophils directly repressed the IL-23/IL-17/G-CSF granulopoietic cytokine cascade. These results provide mechanistic insight into the molecular events orchestrating neutrophil homeostasis and advance our understanding of LXRs as integrators of phagocyte function, lipid metabolism, and cytokine gene expression.
Cynthia Hong, Yoko Kidani, Noelia A-Gonzalez, Tram Phung, Ayaka Ito, Xin Rong, Katrin Ericson, Hanna Mikkola, Simon W. Beaven, Lloyd S. Miller, Wen-Hai Shao, Philip L. Cohen, Antonio Castrillo, Peter Tontonoz, Steven J. Bensinger
Ectodermal dysplasia with immune deficiency (EDI) is an immunological and developmental disorder caused by alterations in the gene encoding NF-κB essential modulator (NEMO; also known as IκB kinase γ subunit [IKKγ]). Missense mutations in the gene encoding NEMO are associated with reduced signal-induced nuclear translocation of NF-κB proteins, resulting in defective expression of NF-κB target genes. Here, we report 2 unrelated male patients with EDI, both of whom have normal NEMO coding sequences, but exhibit a marked reduction in expression of full-length NEMO protein. TLR4 stimulation of APCs from these patients induced normal cytoplasmic activation and nuclear translocation of NF-κB. However, cells deficient in full-length NEMO were defective in expression of NF-κB–regulated cytokines, such as IL-12, suggesting a downstream defect in chromatin accessibility for NF-κB transcription factors. TLR4-stimulated APCs from the patients were defective in IKKα-dependent H3 histone phosphorylation at the IL-12 promoter and recruitment of NF-κB heterodimers RelA and cRel to the promoter. Expression of a super-active form of IKKα restored IL-12 production in a NEMO knockdown human monocytic cell line following LPS treatment. Our findings suggest that NEMO regulates the nuclear function of IKKα and offer new insights into the mechanisms underlying diminished NF-κB signaling in patients with EDI.
Stephane T. Temmerman, Chi A. Ma, Yongge Zhao, Jeffrey Keenan, Ivona Aksentijevich, Margaret Fessler, Margaret R. Brown, Alan Knutsen, Ralph Shapiro, Ashish Jain
It is estimated that one-third of the world’s population is infected with Mycobacterium tuberculosis. Infection typically remains latent, but it can reactivate to cause clinical disease. The only vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), is largely ineffective, and ways to enhance its efficacy are being developed. Of note, the candidate booster vaccines currently under clinical development have been designed to improve BCG efficacy but not prevent reactivation of latent infection. Here, we demonstrate that administering a multistage vaccine that we term H56 in the adjuvant IC31 as a boost to vaccination with BCG delays and reduces clinical disease in cynomolgus macaques challenged with M. tuberculosis and prevents reactivation of latent infection. H56 contains Ag85B and ESAT-6, which are two of the M. tuberculosis antigens secreted in the acute phase of infection, and the nutrient stress–induced antigen Rv2660c. Boosting with H56/IC31 resulted in efficient containment of M. tuberculosis infection and reduced rates of clinical disease, as measured by clinical parameters, inflammatory markers, and improved survival of the animals compared with BCG alone. Boosted animals showed reduced pulmonary pathology and extrapulmonary dissemination, and protection correlated with a strong recall response against ESAT-6 and Rv2660c. Importantly, BCG/H56-vaccinated monkeys did not reactivate latent infection after treatment with anti-TNF antibody. Our results indicate that H56/IC31 boosting is able to control late-stage infection with M. tuberculosis and contain latent tuberculosis, providing a rationale for the clinical development of H56.
Philana Ling Lin, Jes Dietrich, Esterlina Tan, Rodolfo M. Abalos, Jasmin Burgos, Carolyn Bigbee, Matthew Bigbee, Leslie Milk, Hannah P. Gideon, Mark Rodgers, Catherine Cochran, Kristi M. Guinn, David R. Sherman, Edwin Klein, Christopher Janssen, JoAnne L. Flynn, Peter Andersen
IgE-mediated activation of mast cells and basophils underlies allergic diseases such as asthma. Histamine-releasing factor (HRF; also known as translationally controlled tumor protein [TCTP] and fortilin) has been implicated in late-phase allergic reactions (LPRs) and chronic allergic inflammation, but its functions during asthma are not well understood. Here, we identified a subset of IgE and IgG antibodies as HRF-interacting molecules in vitro. HRF was able to dimerize and bind to Igs via interactions of its N-terminal and internal regions with the Fab region of Igs. Therefore, HRF together with HRF-reactive IgE was able to activate mast cells in vitro. In mouse models of asthma and allergy, Ig-interacting HRF peptides that were shown to block HRF/Ig interactions in vitro inhibited IgE/HRF-induced mast cell activation and in vivo cutaneous anaphylaxis and airway inflammation. Intranasally administered HRF recruited inflammatory immune cells to the lung in naive mice in a mast cell– and Fc receptor–dependent manner. These results indicate that HRF has a proinflammatory role in asthma and skin immediate hypersensitivity, leading us to suggest HRF as a potential therapeutic target.
Jun-ichi Kashiwakura, Tomoaki Ando, Kenji Matsumoto, Miho Kimura, Jiro Kitaura, Michael H. Matho, Dirk M. Zajonc, Tomomitsu Ozeki, Chisei Ra, Susan M. MacDonald, Reuben P. Siraganian, David H. Broide, Yuko Kawakami, Toshiaki Kawakami
Herpes simplex encephalitis (HSE) is the most common sporadic viral encephalitis of childhood. Autosomal recessive (AR) UNC-93B and TLR3 deficiencies and autosomal dominant (AD) TLR3 and TRAF3 deficiencies underlie HSE in some children. We report here unrelated HSE children with AR or AD TRIF deficiency. The AR form of the disease was found to be due to a homozygous nonsense mutation that resulted in a complete absence of the TRIF protein. Both the TLR3- and the TRIF-dependent TLR4 signaling pathways were abolished. The AD form of disease was found to be due to a heterozygous missense mutation, resulting in a dysfunctional protein. In this form of the disease, the TLR3 signaling pathway was impaired, whereas the TRIF-dependent TLR4 pathway was unaffected. Both patients, however, showed reduced capacity to respond to stimulation of the DExD/H-box helicases pathway. To date, the TRIF-deficient patients with HSE described herein have suffered from no other infections. Moreover, as observed in patients with other genetic etiologies of HSE, clinical penetrance was found to be incomplete, as some HSV-1–infected TRIF-deficient relatives have not developed HSE. Our results provide what we believe to be the first description of human TRIF deficiency and a new genetic etiology for HSE. They suggest that the TRIF-dependent TLR4 and DExD/H-box helicase pathways are largely redundant in host defense. They further demonstrate the importance of TRIF for the TLR3-dependent production of antiviral IFNs in the CNS during primary infection with HSV-1 in childhood.
Vanessa Sancho-Shimizu, Rebeca Pérez de Diego, Lazaro Lorenzo, Rabih Halwani, Abdullah Alangari, Elisabeth Israelsson, Sylvie Fabrega,, Annabelle Cardon, Jerome Maluenda, Megumi Tatematsu, Farhad Mahvelati, Melina Herman, Michael Ciancanelli, Yiqi Guo, Zobaida AlSum, Nouf Alkhamis, Abdulkarim S. Al-Makadma, Ata Ghadiri, Soraya Boucherit, Sabine Plancoulaine, Capucine Picard, Flore Rozenberg, Marc Tardieu, Pierre Lebon, Emmanuelle Jouanguy, Nima Rezaei, Tsukasa Seya, Misako Matsumoto, Damien Chaussabel, Anne Puel, Shen-Ying Zhang, Laurent Abel, Saleh Al-Muhsen, Jean-Laurent Casanova