Juvenile dermatomyositis (JDM) is a debilitating pediatric autoimmune disease manifesting with characteristic rash and muscle weakness. To delineate signaling abnormalities in JDM, mass cytometry was performed with PBMCs from treatment-naive JDM patients and controls. NK cell percentages were lower while frequencies of naive B cells and naive CD4+ T cells were higher in JDM patients than in controls. These cell frequency differences were attenuated with cessation of active disease. A large number of signaling differences were identified in treatment-naive JDM patients compared with controls. Classification models incorporating feature selection demonstrated that differences in phospholipase Cγ2 (PLCγ2) phosphorylation comprised 10 of 12 features (i.e., phosphoprotein in a specific immune cell subset) distinguishing the 2 groups. Because NK cells represented 5 of these 12 features, further studies focused on the PLCγ2 pathway in NK cells, which is responsible for stimulating calcium flux and cytotoxic granule movement. No differences were detected in upstream signaling or total PLCγ2 protein levels. Hypophosphorylation of PLCγ2 and downstream mitogen-activated protein kinase-activated protein kinase 2 were partially attenuated with cessation of active disease. PLCγ2 hypophosphorylation in treatment-naive JDM patients resulted in decreased calcium flux. The identification of dysregulation of PLCγ2 phosphorylation and decreased calcium flux in NK cells provides potential mechanistic insight into JDM pathogenesis.
Allison A. Throm, Joshua B. Alinger, Jeanette T. Pingel, Allyssa L. Daugherty, Lauren M. Pachman, Anthony R. French
Triple-negative breast cancer (TNBC) has few therapeutic options, and alternative approaches are urgently needed. Stimulator of IFN genes (STING) is becoming an exciting target for therapeutic adjuvants. However, STING resides inside the cell, and the intracellular delivery of CDNs, such as cGAMP, is required for the optimal activation of STING. We show that liposomal nanoparticle-delivered cGAMP (cGAMP-NP) activates STING more effectively than soluble cGAMP. These particles induce innate and adaptive host immune responses to preexisting tumors in both orthotopic and genetically engineered models of basal-like TNBC. cGAMP-NPs also reduce melanoma tumor load, with limited responsivity to anti–PD-L1. Within the tumor microenvironment, cGAMP-NPs direct both mouse and human macrophages (M), reprograming from protumorigenic M2-like phenotype toward M1-like phenotype; enhance MHC and costimulatory molecule expression; reduce M2 biomarkers; increase IFN-γ–producing T cells; augment tumor apoptosis; and increase CD4+ and CD8+ T cell infiltration. Activated T cells are required for tumor suppression, as their depletion reduces antitumor activity. Importantly, cGAMP-NPs prevent the formation of secondary tumors, and a single dose is sufficient to inhibit TNBC. These data suggest that a minimal system comprised of cGAMP-NP alone is sufficient to modulate the tumor microenvironment to effectively control PD-L1–insensitive TNBC.
Ning Cheng, Rebekah Watkins-Schulz, Robert D. Junkins, Clément N. David, Brandon M. Johnson, Stephanie A. Montgomery, Kevin J. Peine, David B. Darr, Hong Yuan, Karen P. McKinnon, Qi Liu, Lei Miao, Leaf Huang, Eric M. Bachelder, Kristy M. Ainslie, Jenny P-Y Ting
Asthma is one of the most common immunological diseases and is characterized by airway hyperresponsiveness (AHR), mucus overproduction, and airway eosinophilia. Although mouse models have provided insight into the mechanisms by which type-2 cytokines induce asthmatic airway inflammation, differences between the rodent and human immune systems hamper efforts to improve understanding of human allergic diseases. In this study, we aim to establish a preclinical animal model of asthmatic airway inflammation using humanized IL-3/GM-CSF or IL-3/GM-CSF/IL-5 Tg NOD/Shi-scid-IL2rγnull (NOG) mice and investigate the roles of human type-2 immune responses in the asthmatic mice. Several important characteristics of asthma — such as AHR, goblet cell hyperplasia, T cell infiltration, IL-13 production, and periostin secretion — were induced in IL-3/GM-CSF Tg mice by intratracheally administered human IL-33. In addition to these characteristics, human eosinophilic inflammation was observed in IL-3/GM-CSF/IL-5 Tg mice. The asthmatic mechanisms of the humanized mice were driven by activation of human Th2 and mast cells by IL-33 stimulation. Furthermore, treatment of the humanized mice with an anti–human IL-13 antibody significantly suppressed these characteristics. Therefore, the humanized mice may enhance our understanding of the pathophysiology of allergic disorders and facilitate the preclinical development of new therapeutics for IL-33–mediated type-2 inflammation in asthma.
Ryoji Ito, Shuichiro Maruoka, Kaori Soda, Ikumi Katano, Kenji Kawai, Mika Yagoto, Asami Hanazawa, Takeshi Takahashi, Tomoyuki Ogura, Motohito Goto, Riichi Takahashi, Shota Toyoshima, Yoshimichi Okayama, Kenji Izuhara, Yasuhiro Gon, Shu Hashimoto, Mamoru Ito, Satoshi Nunomura
Glioblastoma (GBM) remains uniformly lethal, and despite a large accumulation of immune cells in the microenvironment, there is limited antitumor immune response. To overcome these challenges, a comprehensive understanding of GBM systemic immune response during disease progression is required. Here, we integrated multiparameter flow cytometry and mass cytometry TOF (CyTOF) analysis of patient blood to determine changes in the immune system among tumor types and over disease progression. Utilizing flow cytometry analysis in a cohort of 259 patients ranging from benign to malignant primary and metastatic brain tumors, we found that GBM patients had a significant elevation in myeloid-derived suppressor cells (MDSCs) in peripheral blood but not immunosuppressive Tregs. In GBM patient tissue, we found that increased MDSC levels in recurrent GBM portended poor prognosis. CyTOF analysis of peripheral blood from newly diagnosed GBM patients revealed that reduced MDSCs over time were accompanied by a concomitant increase in DCs. GBM patients with extended survival also had reduced MDSCs, similar to the levels of low-grade glioma (LGG) patients. Our findings provide a rationale for developing strategies to target MDSCs, which are elevated in GBM patients and predict poor prognosis.
Tyler J. Alban, Alvaro G. Alvarado, Mia D. Sorensen, Defne Bayik, Josephine Volovetz, Emily Serbinowski, Erin E. Mulkearns-Hubert, Maksim Sinyuk, James S. Hale, Giovana R. Onzi, Mary McGraw, Pengjing Huang, Matthew M. Grabowski, Connor A. Wathen, Manmeet S. Ahluwalia, Tomas Radivoyevitch, Harley I. Kornblum, Bjarne W. Kristensen, Michael A. Vogelbaum, Justin D. Lathia
Cancer incidence increases with age, but paradoxically, cancers have been found to grow more quickly in young mice compared with aged ones. The cause of differential tumor growth has been debated and, over time, attributed to faster tumor cell proliferation, decreased tumor cell apoptosis, and/or increased angiogenesis in young animals. Despite major advances in our understanding of tumor immunity over the past 2 decades, little attention has been paid to comparing immune cell populations in young and aged mice. Using mouse colon adenocarcinoma model MC38 implanted in young and mature mice, we show that age substantially influences the number of tumor-infiltrating cytotoxic CD8+ T cells, which control cancer progression. The different tumor growth pace in young and mature mice was abrogated in RAG1null mice, which lack mature T and B lymphocytes, and upon selective depletion of endogenous CD8+ cells. Transcriptome analysis further indicated that young mice have decreased levels of the Itga4 gene (CD49d, VLA-4) in tumor-infiltrating lymphocytes when compared with mature mice. Hypothesizing that VLA-4 can have a tumor-protective effect, we depleted the protein, which resulted in accelerated tumor growth in mature mice. These observations may explain the paradoxical growth rates observed in murine cancers, point to the central role of VLA-4 in controlling tumor growth, and open new venues to therapeutic manipulation.
Juhyun Oh, Angela Magnuson, Christophe Benoist, Mikael J. Pittet, Ralph Weissleder
BACKGROUND. Our understanding of phenotypic and functional signatures of CD8+ T cell dysfunction in acute myeloid leukemia (AML) is limited. Deciphering these deranged T cell functional states and how they are impacted by induction chemotherapy is essential for incorporation of novel immune-based strategies to restore and maintain antileukemia immunity. METHODS. We utilized high-dimensional immunophenotyping, gene expression, and functional studies to characterize peripheral blood and bone marrow CD8+ T cells in 72 AML patients at diagnosis and after induction chemotherapy. RESULTS. Our data suggest that multiple aspects of deranged T cell function are operative in AML at diagnosis, with exhaustion and senescence being the dominant processes. Following treatment, the phenotypic and transcriptional profile of CD8+ T cells diverged between responders and nonresponders. Response to therapy correlated with upregulation of costimulatory, and downregulation of apoptotic and inhibitory, T cell signaling pathways, indicative of restoration of T cell function. In functional studies, AML blasts directly altered CD8+ T cell viability, expansion, co-signaling and senescence marker expression. This CD8+ T cell dysfunction was in part reversible upon PD-1 blockade or OX40 costimulation in vitro. CONCLUSION. Our findings highlight the uniqueness of AML in sculpting CD8+ T cell responses and the plasticity of their signatures upon chemotherapy response, providing a compelling rationale for integration of novel immunotherapies to augment antileukemia immunity. FUNDING. This work was supported by the Leukemia & Lymphoma Society grant no. 6449-13; NIH grants UM1-CA186691 and R01-HL110907-01; the American Society for Blood and Marrow Transplantation New Investigator Award/Gabrielle’s Angel Foundation; the Vienna Fund for Innovative Cancer Research; and by fellowships from the Wenner-Gren Foundation and the Swedish Society for Medical Research.
Hanna A. Knaus, Sofia Berglund, Hubert Hackl, Amanda L. Blackford, Joshua F. Zeidner, Raúl Montiel-Esparza, Rupkatha Mukhopadhyay, Katrina Vanura, Bruce R. Blazar, Judith E. Karp, Leo Luznik, Ivana Gojo
BACKGROUND. The clearance of hepatitis B surface antigen (HBsAg) loss, defined as functional cure, is a clinical target in patients with chronic hepatitis B (CH). To understand the immune responses underlying functional cure, we evaluated cytokine and chemokine expression profiles from patients with resolving and nonresolving acute hepatitis B (AH). METHODS. We cross-sectionally evaluated 41 chemokines and cytokines at the peak of hepatitis in the sera from 41 self-limited AH patients who achieved HBsAg seroconversion, 8 AH patients who failed to clear HBsAg within 1 year after the diagnosis, 8 CH patients with hepatic flare, and 14 healthy volunteers. We longitudinally examined 41 chemokines and cytokines in the sera from 4 self-limited AH patients, 3 chimpanzees inoculated with hepatitis B virus (HBV), and 2 CH patients treated with nucleotide analogs and PEG–IFN-α, one resulting in functional cure. RESULTS. In AH patients and HBV-inoculated chimpanzees with HBsAg loss, CXCL9, CXCL10, CXCL11, CXCL13, and IL-21 were elevated at hepatitis with subsequent decline of HBsAg. Interestingly, IL-21 elevation was observed only in resolving AH patients but not in nonresolvers. CXCL13 and IL-21 elevation was not observed in CH patients who failed to attain HBsAg loss, even at hepatic flare. A concomitant increase of CXCL13 and IL-21 was significant in CH patients who attained HBsAg seroconversion with a sequential therapy. CONCLUSION. Elevation of serum CXCL9, CXCL10, CXCL11, CXCL13, and IL-21 might be a hallmark of functional cure of AH or CH patients.
Sachiyo Yoshio, Yohei Mano, Hiroyoshi Doi, Hirotaka Shoji, Tomonari Shimagaki, Yuzuru Sakamoto, Hironari Kawai, Michitaka Matsuda, Taizo Mori, Yosuke Osawa, Masaaki Korenaga, Masaya Sugiyama, Masashi Mizokami, Eiji Mita, Keiko Katayama, Junko Tanaka, Tatsuya Kanto
Posttransplant cyclophosphamide (PTCy) has been found to be effective in ameliorating acute graft-versus-host disease (GVHD) in patients following allogeneic hematopoietic stem cell transplantation (aHSCT). Adoptive transfer of high numbers of donor Tregs in experimental aHSCT has shown promise as a therapeutic modality for GVHD regulation. We recently described a strategy for in vivo Treg expansion targeting two receptors: TNFRSF25 and CD25. To date, there have been no direct comparisons between the use of PTCy and Tregs regarding outcome and immune reconstitution within identical groups of transplanted mice. Here, we assessed these two strategies and found both decreased clinical GVHD and improved survival long term. However, recipients transplanted with Treg-expanded donor cells (TrED) exhibited less weight loss early after HSCT. Additionally, TrED recipients demonstrated less thymic damage, significantly more recent thymic emigrants, and more rapid lymphoid engraftment. Three months after HSCT, PTCy-treated and TrED recipients showed tolerance to F1 skin allografts and comparable immune function. Overall, TrED was found superior to PTCy with regard to weight loss early after transplant and initial lymphoid engraftment. Based on these findings, we speculate that morbidity and mortality after transplant could be diminished following TrED transplant into aHSCT recipients, and, therefore, that TrED could provide a promising clinical strategy for GVHD prophylaxis.
Dietlinde Wolf, Cameron S. Bader, Henry Barreras, Sabrina Copsel, Brent J. Pfeiffer, Casey O. Lightbourn, Norman H. Altman, Krishna V. Komanduri, Robert B. Levy
Recent studies in cancer research have focused intensely on the antineoplastic effects of immune checkpoint inhibitors. While the development of these inhibitors has progressed successfully, strategies to further improve their efficacy and reduce their toxicity are still needed. We hypothesized that the delivery of anti–PD-1 antibody encapsulated in PLGA nanoparticles (anti–PD-1 NPs) to the spleen would improve the antitumor effect of this agent. Unexpectedly, we found that mice treated with a high dose of anti–PD-1 NPs exhibited significantly higher mortality compared with those treated with free anti–PD-1 antibody, due to the overactivation of T cells. Administration of anti–PD-1 NPs to splenectomized LT-α–/– mice, which lack both lymph nodes and spleen, resulted in a complete reversal of this increased mortality and revealed the importance of secondary lymphoid tissues in mediating anti–PD-1–associated toxicity. Attenuation of the anti–PD-1 NPs dosage prevented toxicity and significantly improved its antitumor effect in the B16-F10 murine melanoma model. Furthermore, we found that anti–PD-1 NPs undergo internalization by DCs in the spleen, leading to their maturation and the subsequent activation of T cells. Our findings provide important clues that can lead to the development of strategies to enhance the efficacy of immune checkpoint inhibitors.
Farideh Ordikhani, Mayuko Uehara, Vivek Kasinath, Li Dai, Siawosh K. Eskandari, Baharak Bahmani, Merve Yonar, Jamil R. Azzi, Yousef Haik, Peter T. Sage, George F. Murphy, Nasim Annabi, Tobias Schatton, Indira Guleria, Reza Abdi
TGF-β is a promising immunotherapeutic target. It is expressed ubiquitously in a latent form that must be activated to function. Determination of where and how latent TGF-β (L-TGF-β) is activated in the tumor microenvironment could facilitate cell- and mechanism-specific approaches to immunotherapeutically target TGF-β. Binding of L-TGF-β to integrin αvβ8 results in activation of TGF-β. We engineered and used αvβ8 antibodies optimized for blocking or detection, which — respectively — inhibit tumor growth in syngeneic tumor models or sensitively and specifically detect β8 in human tumors. Inhibition of αvβ8 potentiates cytotoxic T cell responses and recruitment of immune cells to tumor centers — effects that are independent of PD-1/PD-L1. β8 is expressed on the cell surface at high levels by tumor cells, not immune cells, while the reverse is true of L-TGF-β, suggesting that tumor cell αvβ8 serves as a platform for activating cell-surface L-TGF-β presented by immune cells. Transcriptome analysis of tumor-associated lymphoid cells reveals macrophages as a key cell type responsive to β8 inhibition with major increases in chemokine and tumor-eliminating genes. High β8 expression in tumor cells is seen in 20%–80% of various cancers, which rarely coincides with high PD-L1 expression. These data suggest tumor cell αvβ8 is a PD-1/PD-L1–independent immunotherapeutic target.
Naoki Takasaka, Robert I. Seed, Anthony Cormier, Andrew J. Bondesson, Jianlong Lou, Ahmed Elattma, Saburo Ito, Haruhiko Yanagisawa, Mitsuo Hashimoto, Royce Ma, Michelle D. Levine, Jean Publicover, Rashaun Potts, Jillian M. Jespersen, Melody G. Campbell, Fraser Conrad, James D. Marks, Yifan Cheng, Jody L. Baron, Stephen L. Nishimura
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