Uncontrolled secretion of type I IFN, as the result of endosomal TLR (i.e., TLR7 and TLR9) signaling in plasmacytoid DCs (pDCs), and abnormal production of autoantibodies by B cells are critical for systemic lupus erythematosus (SLE) pathogenesis. The importance of galectin-9 (Gal-9) in regulating various autoimmune diseases, including lupus, has been demonstrated. However, the precise mechanism by which Gal-9 mediates this effect remains unclear. Here, using spontaneous murine models of lupus (i.e., BXSB/MpJ and NZB/W F1 mice), we demonstrate that administration of Gal-9 results in reduced TLR7-mediated autoimmune manifestations. While investigating the mechanism underlying this phenomenon, we observed that Gal-9 inhibits the phenotypic maturation of pDCs and B cells and abrogates their ability to mount cytokine responses to TLR7/TLR9 ligands. Importantly, immunocomplex-mediated (IC-mediated) and neutrophil extracellular trap–mediated (NET-mediated) pDC activation was inhibited by Gal-9. Additionally, the mTOR/p70S6K pathway, which is recruited by both pDCs and B cells for TLR-mediated IFN secretion and autoantibody generation, respectively, was attenuated. Gal-9 was found to exert its inhibitory effect on both the cells by interacting with CD44.
Santosh K. Panda, Valeria Facchinetti, Elisaveta Voynova, Shino Hanabuchi, Jodi L. Karnell, Richard N. Hanna, Roland Kolbeck, Miguel A. Sanjuan, Rachel Ettinger, Yong-Jun Liu
In situ cancer vaccines are under active clinical investigation due to their reported ability to eradicate both local and disseminated malignancies. Intratumoral vaccine administration is thought to activate a T cell mediated immune response, which begins in the treated tumor and cascades systemically. We describe a positron emission tomography tracer (64Cu-DOTA-AbOX40) that enabled non-invasive and longitudinal imaging of OX40, a cell surface marker of T cell activation. We report the spatiotemporal dynamics of T cell activation following in situ vaccination with CpG oligodeoxynucleotide, in a dual tumor bearing mouse model. We demonstrate that OX40 imaging could predict tumor responses at day 9 post treatment based on tumor tracer uptake at day 2, with higher accuracy than both anatomical and blood-based measurements. These studies provide key insights into global T cell activation following local CpG treatment and indicate that 64Cu-DOTA-AbOX40 is a promising candidate for monitoring clinical cancer immunotherapy strategies.
Israt S. Alam, Aaron T. Mayer, Idit Sagiv-Barfi, Kezheng Wang, Ophir Vermesh, Debra K. Czerwinski, Emily M. Johnson, Michelle L. James, Ronald Levy, Sanjiv S. Gambhir
Gout is the most common inflammatory arthritis affecting men. Acute gouty inflammation is triggered by monosodium urate (MSU) crystal deposition in and around joints that activates macrophages into a proinflammatory state, resulting in neutrophil recruitment. A complete understanding of how MSU crystals activate macrophages in vivo has been difficult because of limitations of live imaging this process in traditional animal models. By live imaging the macrophage and neutrophil response to MSU crystals within an intact host (larval zebrafish), we reveal that macrophage activation requires mitochondrial ROS (mROS) generated through fatty acid oxidation. This mitochondrial source of ROS contributes to NF-κB–driven production of IL-1β and TNF-α, which promote neutrophil recruitment. We demonstrate the therapeutic utility of this discovery by showing that this mechanism is conserved in human macrophages and, via pharmacologic blockade, that it contributes to neutrophil recruitment in a mouse model of acute gouty inflammation. To our knowledge, this study is the first to uncover an immunometabolic mechanism of macrophage activation that operates during acute gouty inflammation. Targeting this pathway holds promise in the management of gout and, potentially, other macrophage-driven diseases.
Christopher J. Hall, Leslie E. Sanderson, Lisa M. Lawrence, Bregina Pool, Maarten van der Kroef, Elina Ashimbayeva, Denver Britto, Jacquie L. Harper, Graham J. Lieschke, Jonathan W. Astin, Kathryn E. Crosier, Nicola Dalbeth, Philip S. Crosier
Emerging data suggest that hypercholesterolemia has stimulatory effects on adaptive immunity and that these effects can promote atherosclerosis and perhaps other inflammatory diseases. However, research in this area has relied primarily on inbred strains of mice, whose adaptive immune system can differ substantially from that of humans. Moreover, the genetically induced hypercholesterolemia in these models typically results in plasma cholesterol levels that are much higher than those in most humans. To overcome these obstacles, we studied human immune system-reconstituted mice (hu-mice) rendered hypercholesterolemic by treatment with AAV8- PCSK9 and a high-fat/high-cholesterol Western-type diet (WD). These mice had a high percentage of human T cells and moderate hypercholesterolemia. Compared with hu-mice having lower plasma cholesterol, the PCSK9-WD mice developed a T cell-mediated inflammatory response in the lung and liver. Human CD4+ and CD8+ T cells bearing an effector memory phenotype were significantly elevated in the blood, spleen, and lungs of PCSK9-WD hu-mice, while splenic and circulating regulatory T cells were reduced. These data show that moderately high plasma cholesterol can disrupt human T cell homeostasis in vivo. This process may not only exacerbate atherosclerosis but also contribute to T cell-mediated inflammatory diseases in the setting of hypercholesterolemia.
Jonathan D. Proto, Amanda C. Doran, Manikandan Subramanian, Hui Wang, Mingyou Zhang, Erdi Sozen, Christina Rymond, George Kuriakose, Vivette D'Agati, Robert Winchester, Megan Sykes, Yong-Guang Yang, Ira Tabas
Despite significant advances in the treatment of multiple myeloma (MM), most patients succumb to disease progression. One of the major immunosuppressive mechanisms that is believed to play a role in myeloma progression, is the expansion of regulatory T-cells (Tregs). In this study, we demonstrate that myeloma cells drive Treg expansion and activation by secreting type-1 interferon (IFN). Blocking IFNAR1 (interferon alpha and beta receptor 1) on Tregs significantly decreases both, myeloma-associated Treg immunosuppressive function and myeloma progression. Using syngeneic transplantable murine myeloma models and bone marrow (BM) aspirates of multiple myeloma patients, we found that Tregs were expanded and activated in the BM microenvironment at early stages of myeloma development. Selective depletion of Tregs led to a complete remission and prolonged survival in mice injected with myeloma cells. Further analysis of the interaction between myeloma cells and Tregs using gene sequencing and enrichment analysis uncovered a feedback loop, wherein myeloma-cell-secreted type-1 IFN induced proliferation and expansion of Tregs. By using IFNAR1-blocking antibody treatment and IFNAR1 knockout Tregs, we demonstrated a significant decrease in myeloma-associated Treg proliferation, which was associated with longer survival of myeloma-injected mice. Our results thus suggest that blocking type-1 IFN signaling represents a potential strategy to target immunosuppressive Treg function in MM.
Yawara Kawano, Oksana Zavidij, Jihye Park, Michele Moschetta, Katsutoshi Kokubun, Tarek H. Mouhieddine, Salomon Manier, Yuji Mishima, Naoka Murakami, Mark Bustoros, Romanos Sklavenitis Pistofidis, Mairead Reidy, Yu J. Shen, Mahshid Rahmat, Pavlo Lukyanchykov, Esilida Sula Karreci, Shokichi Tsukamoto, Jiantao Shi, Satoshi Takagi, Daisy Huynh, Antonio Sacco, Yu-Tzu Tai, Marta Chesi, P. Leif Bergsagel, Aldo M. Roccaro, Jamil Azzi, Irene M. Ghobrial
Apoptosis has been proposed as a key mechanism responsible for CD4+ T cell depletion and immune dysfunction during HIV infection. We demonstrated that Q-VD-OPH, a caspase inhibitor, inhibits spontaneous and activation-induced death of T cells from SIV-infected rhesus macaques (RMs). When administered during the acute phase of infection, Q-VD-OPH was associated with (a) reduced levels of T cell death, (b) preservation of CD4+/CD8+ T cell ratio in lymphoid organs and in the gut, (c) maintenance of memory CD4+ T cells, and (d) increased specific CD4+ T cell response associated with the expression of cytotoxic molecules. Although therapy was limited to the acute phase of infection, Q-VD-OPH–treated RMs showed lower levels of both viral load and cell-associated SIV DNA as compared with control SIV-infected RMs throughout the chronic phase of infection, and prevented the development of AIDS. Overall, our data demonstrate that Q-VD-OPH injection in SIV-infected RMs may represent an adjunctive therapeutic agent to control HIV infection and delaying disease progression to AIDS.
Mireille Laforge, Ricardo Silvestre, Vasco Rodrigues, Julie Garibal, Laure Campillo-Gimenez, Shahul Mouhamad, Valérie Monceaux, Marie-Christine Cumont, Henintsoa Rabezanahary, Alain Pruvost, Anabela Cordeiro-da-Silva, Bruno Hurtrel, Guido Silvestri, Anna Senik, Jérôme Estaquier
Polypeptide vaccines effectively activate human T cells but suffer from poor biological stability, which confines both transport logistics and in vivo therapeutic activity. Synthetic biology has the potential to address these limitations through the generation of highly stable antigenic “mimics” using subunits that do not exist in the natural world. We developed a platform based on D–amino acid combinatorial chemistry and used this platform to reverse engineer a fully artificial CD8+ T cell agonist that mirrored the immunogenicity profile of a native epitope blueprint from influenza virus. This nonnatural peptide was highly stable in human serum and gastric acid, reflecting an intrinsic resistance to physical and enzymatic degradation. In vitro, the synthetic agonist stimulated and expanded an archetypal repertoire of polyfunctional human influenza virus–specific CD8+ T cells. In vivo, specific responses were elicited in naive humanized mice by subcutaneous vaccination, conferring protection from subsequent lethal influenza challenge. Moreover, the synthetic agonist was immunogenic after oral administration. This proof-of-concept study highlights the power of synthetic biology to expand the horizons of vaccine design and therapeutic delivery.
John J. Miles, Mai Ping Tan, Garry Dolton, Emily S.J. Edwards, Sarah A.E. Galloway, Bruno Laugel, Mathew Clement, Julia Makinde, Kristin Ladell, Katherine K. Matthews, Thomas S. Watkins, Katie Tungatt, Yide Wong, Han Siean Lee, Richard J. Clark, Johanne M. Pentier, Meriem Attaf, Anya Lissina, Ann Ager, Awen Gallimore, Pierre J. Rizkallah, Stephanie Gras, Jamie Rossjohn, Scott R. Burrows, David K. Cole, David A. Price, Andrew K. Sewell
UL18 is a human CMV (HCMV) MHC class I (MHCI) homolog that efficiently inhibits leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1)+ NK cells. We found an association of LILRB1 polymorphisms in the regulatory regions and ligand-binding domains with control of HCMV in transplant patients. Naturally occurring LILRB1 variants expressed in model NK cells showed functional differences with UL18 and classical MHCI, but not with HLA-G. The altered functional recognition was recapitulated in binding assays with the binding domains of LILRB1. Each of 4 nonsynonymous substitutions in the first 2 LILRB1 immunoglobulin domains contributed to binding with UL18, classical MHCI, and HLA-G. One of the polymorphisms controlled addition of an N-linked glycan, and that mutation of the glycosylation site altered binding to all ligands tested, including enhancing binding to UL18. Together, these findings indicate that specific LILRB1 alleles that allow for superior immune evasion by HCMV are restricted by mutations that limit LILRB1 expression selectively on NK cells. The polymorphisms also maintained an appropriate interaction with HLA-G, fitting with a principal role of LILRB1 in fetal tolerance.
Kang Yu, Chelsea L. Davidson, Agnieszka Wójtowicz, Luiz Lisboa, Ting Wang, Adriana M. Airo, Jean Villard, Jeremie Buratto, Tatyana Sandalova, Adnane Achour, Atul Humar, Katia Boggian, Alexia Cusini, Christian van Delden, Adrian Egli, Oriol Manuel, Nicolas Mueller, Pierre-Yves Bochud, Swiss Transplant Cohort Study, Deborah N. Burshtyn
Intralesional therapy with oncolytic viruses (OVs) leads to the activation of local and systemic immune pathways, which may present targets for further combinatorial therapies. Here, we used human tumor histocultures as well as syngeneic tumor models treated with Newcastle disease virus (NDV) to identify a range of immune targets upregulated with OV treatment. Despite tumor infiltration of effector T lymphocytes in response to NDV, there was ongoing inhibition through programmed death ligand 1 (PD-L1), acting as a mechanism of early and late adaptive immune resistance to the type I IFN response and T cell infiltration, respectively. Systemic therapeutic targeting of programmed cell death receptor 1 (PD-1) or PD-L1 in combination with intratumoral NDV resulted in the rejection of both treated and distant tumors. These findings have implications for the timing of PD-1/PD-L1 blockade in conjunction with OV therapy and highlight the importance of understanding the adaptive mechanisms of immune resistance to specific OVs for the rational design of combinatorial approaches using these agents.
Dmitriy Zamarin, Jacob M. Ricca, Svetlana Sadekova, Anton Oseledchyk, Ying Yu, Wendy M. Blumenschein, Jerelyn Wong, Mathieu Gigoux, Taha Merghoub, Jedd D. Wolchok
Non-antigen-specific stimulatory cancer immunotherapies are commonly complicated by off-target effects. Antigen-specific immunotherapy, combining viral tumor antigen or personalised neo-epitopes with immune targeting, offers a solution. However, the lack of flexible systems targeting tumor antigens to cross-presenting dendritic cells (DCs) limits clinical development. Although antigen-anti-CLEC-9A mAb conjugates target cross-presenting DCs, adjuvant must be co-delivered for cytotoxic T-cell (CTL) induction. We functionalized tailored nanoemulsions encapsulating tumor antigens to target Clec9A (Clec9A-TNE). Clec9A-TNE encapsulating ovalbumin (OVA) antigen targeted and activated cross-presenting DCs without additional adjuvant, promoting antigen-specific CD4+ and CD8+ T cell proliferation, CTL and antibody responses. OVA-Clec9A-TNE-induced DC activation required CD4 and CD8 epitopes, CD40 and IFN-α. Clec9A-TNE encapsulating human papillomavirus (HPV) E6-E7 significantly suppressed HPV-associated tumor growth while E6-E7-CpG did not. Clec9A-TNE loaded with pooled B16F10 melanoma neo-epitopes induced epitope-specific CD4+ and CD8+ T cell responses, permitting selection of immunogenic neo-epitopes. Clec9A-TNE encapsulating six neo-epitopes significantly suppressed B16-F10 melanoma growth in a CD4 T cell-dependent manner. Thus, cross-presenting DCs targeted with antigen-Clec9A-TNE stimulate therapeutically-effective tumor-specific immunity, dependent on T cell help.
Bijun Zeng, Anton P.J. Middelberg, Adrian Gemiarto, Kelli MacDonald, Alan G. Baxter, Meghna Talekar, Davide Moi, Kirsteen M. Tullett, Irina Caminschi, Mireille H. Lahoud, Roberta Mazzieri, Riccardo Dolcetti, Ranjeny Thomas