Recovery from measles results in life-long protective immunity. To understand induction of long-term immunity, rhesus macaques were studied for six months after infection with WT measles virus (MeV). Infection caused viremia and rash with clearance of infectious virus by 14 days. MeV RNA persisted in PBMCs for 30-90 days and in lymphoid tissue for 6 months most often in B cells but was rarely detected in BM. Antibody with neutralizing activity and binding specificity for MeV nucleocapsid (N), hemagglutinin (H) and fusion proteins appeared with the rash and avidity matured over 3-4 months. Lymph nodes had increasing numbers of MeV-specific antibody-secreting cells (ASCs) and germinal centers with late hyalinization. ASCs appeared in circulation with the rash and continued to appear along with peripheral Tfh cells for the study duration. ASCs in lymph nodes and PBMCs produced antibody to both H and N, with more H-specific ASCs in BM. From 14-21 days 20-100-fold more total ASCs than MeV-specific ASCs appeared in circulation suggesting mobilization of pre-existing ASCs. Therefore, persistence of MeV RNA in lymphoid tissue was accompanied by continued germinal center formation, ASC production, avidity maturation and accumulation of H-specific ASCs in BM to sustain neutralizing antibody and protective immunity.
Ashley N. Nelson, Wen-Hsuan W. Lin, Rupak Shivakoti, Nicole E. Putnam, Lisa M. Mangus, Robert J. Adams, Debra Hauer, Victoria K. Baxter, Diane E. Griffin
Dengue (DENV) and Zika viruses (ZIKV) are closely related mosquito-borne flaviviruses that co-circulate in tropical regions and constitute major threats to global human health. Whether preexisting immunity to one virus affects disease caused by the other during primary or secondary infections is unknown but is critical in preparing for future outbreaks and predicting vaccine safety. Using a human skin explant model, we show that DENV-3 immune sera increased recruitment and infection of Langerhans cells, macrophages and dermal dendritic cells following inoculation with DENV-2 or ZIKV. Similarly, ZIKV immune sera enhanced infection with DENV-2. Immune sera increased migration of infected Langerhans cells to dermis and emigration of infected cells out of skin. Heterotypic immune sera increased viral RNA in dermis almost tenfold and reduced the amount of virus required to infect a majority of myeloid cells by 100 to 1,000 fold. Enhancement was associated with cross-reactive IgG and induction of IL-10 expression and was mediated by both CD32 and CD64 Fcγ receptors. These findings reveal that preexisting heterotypic immunity greatly enhances DENV and ZIKV infection, replication and spread in human skin. This relevant tissue model will be valuable in assessing the efficacy and risk of dengue and Zika vaccines in humans.
Priscila M.S. Castanha, Geza Erdos, Simon C. Watkins, Louis D. Falo, Jr., Ernesto T.A. Marques, Simon M. Barratt-Boyes
Genetic variants within/near the interferon regulatory factor 5 (IRF5) locus associate with systemic lupus erythematosus (SLE) across ancestral groups. The major IRF5-SLE risk haplotype is common across populations, yet immune functions for the risk haplotype are undefined. We characterized the global immune-phenotype of healthy donors homozygous for the major risk and non-risk haplotypes and identified cell lineage-specific alterations that mimic pre-symptomatic SLE. Contrary to previous studies in B lymphoblastoid cell lines and SLE immune cells, IRF5 genetic variants had little effect on IRF5 protein levels in healthy donors. Instead, we detected basal IRF5 hyper-activation in the myeloid compartment of risk donors that drives the SLE immune-phenotype. Risk donors were ANA positive with anti-Ro and -MPO specificity, had increased circulating plasma cells and plasmacytoid dendritic cells, and enhanced spontaneous NETosis. The IRF5-SLE immune-phenotype was conserved over time and probed mechanistically by ex vivo co-culture, indicating that risk neutrophils are drivers of the global immune-phenotype. RNA-seq of risk neutrophils revealed increased IRF5 transcript expression, IFN pathway enrichment and decreased expression of ROS pathway genes. Altogether, data support that individuals carrying the IRF5-SLE risk haplotype are more susceptible to environmental/stochastic influences that trigger chronic immune activation, predisposing to the development of clinical SLE.
Dan Li, Bharati Matta, Su Song, Victoria Nelson, Kirsten Diggins, Kim R. Simpfendorfer, Peter K. Gregersen, Peter Linsley, Betsy J. Barnes
A recent study of AHSCT for active relapsing-remitting multiple sclerosis (RRMS) showed efficacy in preventing disease worsening. However, the immunologic basis for efficacy remains poorly defined. MS pathology is known to be driven by inflammatory T cells that infiltrate the central nervous system (CNS). Therefore, we hypothesized that the pre-existing T cell repertoire in the intrathecal compartment of active RRMS participants was ablated, and replaced with new clones following AHSCT. T cell repertoires were assessed using high-throughput TCRβ chain sequencing in paired cerebrospinal fluid (CSF) and peripheral blood CD4+ and CD8+ T cells from participants that underwent AHSCT, before and up to 4 years following transplantation. >90% of the pre-existing CSF repertoire in participants with active RRMS was removed following AHSCT, and replaced with clonotypes predominantly generated from engrafted autologous stem cells. Of the pre-existing clones in CSF, ~60% were also detected in pre-therapy blood, and concordant treatment effects were observed for clonotypes in both compartments following AHSCT. These results indicate that replacement of the pre-existing TCR repertoire in active RRMS is a mechanism for AHSCT efficacy, and suggest that peripheral blood could serve as a surrogate for CSF to define mechanisms associated with efficacy in future studies of AHSCT.
Kristina M. Harris, Noha Lim, Paul Lindau, Harlan Robins, Linda M. Griffith, Richard A. Nash, Laurence A. Turka, Paolo A. Muraro
Anti-PD1 therapy has become an immunotherapeutic backbone for treating many cancer types. While many studies have aimed to characterize the immune response to anti-PD1 therapy in the tumor and in the peripheral blood, relatively less is known about the changes in the tumor draining lymph nodes (TDLNs). TDLNs are primary sites of tumor antigen exposure that are critical to both regulation and cross-priming of the antitumor immune response. We employed multi-panel mass cytometry to obtain a high-parameter proteomic (39 total unique markers) immune profile of the TDLN in a well-studied PD1-responsive immunocompetent mouse model. Based on combined hierarchal gating and unsupervised clustering analyses, we found that anti-PD1 therapy enhances remodeling of both B and T cell compartments toward memory phenotypes. Functionally, expression of checkpoint markers was increased in conjunction with production of IFNγ, TNFα, or IL2 in key cell types, including B and T cell subtypes and rarer subsets such as Tregs and NKT cells. A deeper profiling of the immunologic changes that occur in the TDLN milieu during effective anti-PD1 therapy may lead to the discovery of novel biomarkers for monitoring response and provide key insights toward developing combination immunotherapeutic strategies.
Won Jin Ho, Mark Yarchoan, Soren Charmsaz, Rebecca M. Munday, Ludmila Danilova, Marcelo B. Sztein, Elana J. Fertig, Elizabeth M. Jaffee
Background. Current clinical biomarkers for the PD-1 blockade therapy are insufficient because they rely only on the tumor properties such as PD-L1 expression frequency and the amount of tumor mutation burden. Identifying reliable responsive biomarkers based on the host immunity is necessary to improve the predictive values. Methods. We investigated the levels of plasma metabolites and T cell properties including energy metabolism markers in the blood of patients with non-small cell lung cancer before and after treatment with nivolumab (n = 55). Predictive value of combination markers statistically selected were evaluated by cross validation and linear discriminant analysis on discovery and validation cohorts, respectively. Correlation between plasma metabolites and T cell markers were investigated. Results. The four metabolites derived from microbiome (hippuric acid), fatty acid oxidation (butyrylcarnitine) and redox (cystine and glutathione disulfide) provided high response probability (AUC = 0.91). Similarly, a combination of four T cell markers, those related to mitochondrial activation (PGC-1 expression and reactive oxygen species), and the frequencies of CD8+ PD-1high and CD4+ T cells demonstrated even higher prediction value (AUC = 0.96). Among the pool of all selected markers, the four T cell markers were exclusively selected as the highest predictive combination probably due to their linkage to the above mentioned metabolite markers. In a prospective validation set (n = 24) these four cellular markers showed a high accuracy rate for the clinical responses of the patients (AUC = 0.92). Conclusion. Combination of biomarkers reflecting host immune activity is quite valuable for the responder prediction.
Ryusuke Hatae, Kenji Chamoto, Young Hak Kim, Kazuhiro Sonomura, Kei Taneishi, Shuji Kawaguchi, Hironori Yoshida, Hiroaki Ozasa, Yuichi Sakamori, Maryam Akrami, Sidonia Fagarasan, Izuru Masuda, Yasushi Okuno, Fumihiko Matsuda, Toyohiro Hirai, Tasuku Honjo
Multiple sclerosis (MS) is an autoimmune neuroinflammatory disease where the underlying mechanisms driving disease progression have remained unresolved. HLA-DR2b (DRB1*15:01) is the most common genetic risk factor for MS. Additionally, TNF and its receptors TNFR1 and TNFR2 play key roles in MS and its preclinical animal model, experimental autoimmune encephalomyelitis (EAE). TNFR2 is believed to ameliorate CNS pathology by promoting remyelination and Treg function. Here, we show that transgenic mice expressing the human MHC class II (MHC-II) allele HLA-DR2b and lacking mouse MHC-II and TNFR2 molecules, herein called DR2bΔR2, developed progressive EAE, while disease was not progressive in DR2b littermates. Mechanistically, expression of the HLA-DR2b favored Th17 cell development, whereas T cell–independent TNFR2 expression was critical for restraining of an astrogliosis-induced proinflammatory milieu and Th17 cell responses, while promoting remyelination. Our data suggest the TNFR2 signaling pathway as a potentially novel mechanism for curtailing astrogliosis and promoting remyelination, thus providing new insights into mechanisms limiting progressive MS.
Itay Raphael, Francisco Gomez-Rivera, Rebecca A. Raphael, Rachel R. Robinson, Saisha Nalawade, Thomas G. Forsthuber
Immune checkpoint inhibitor (ICI) treatment has recently become a first-line therapy for many non–small cell lung cancer (NSCLC) patients. Unfortunately, most NSCLC patients are refractory to ICI monotherapy, and initial attempts to address this issue with secondary therapeutics have proven unsuccessful. To identify entities precluding CD8+ T cell accumulation in this process, we performed unbiased analyses on flow cytometry, gene expression, and multiplexed immunohistochemical data from a NSCLC patient cohort. The results revealed the presence of a myeloid-rich subgroup, which was devoid of CD4+ and CD8+ T cells. Of all myeloid cell types assessed, neutrophils were the most highly associated with the myeloid phenotype. Additionally, the ratio of CD8+ T cells to neutrophils (CD8/PMN) within the tumor mass optimally distinguished between active and myeloid cases. This ratio was also capable of showing the separation of patients responsive to ICI therapy from those with stable or progressive disease in 2 independent cohorts. Tumor-bearing mice treated with a combination of anti-PD1 and SX-682 (CXCR1/2 inhibitor) displayed relocation of lymphocytes from the tumor periphery into a malignant tumor, which was associated with induction of IFN-γ–responsive genes. These results suggest that neutrophil antagonism may represent a viable secondary therapeutic strategy to enhance ICI treatment outcomes.
Julia Kargl, Xiaodong Zhu, Huajia Zhang, Grace H. Y. Yang, Travis J. Friesen, Melissa Shipley, Dean Y. Maeda, John A. Zebala, Jill McKay-Fleisch, Gavin Meredith, Afshin Mashadi-Hossein, Christina Baik, Robert H. Pierce, Mary W. Redman, Jeffrey C. Thompson, Steven M. Albelda, Hamid Bolouri, A. McGarry Houghton
BACKGROUND. The cytokine IL-7 is critical for T cell development and function. We performed a Phase Ib study in patients with type 1 diabetes (T1D) to evaluate how blockade of IL-7 would affect immune cells and relevant clinical responses. METHODS. Thirty-seven subjects with T1D received s.c. RN168, a monoclonal antibody that blocks the IL -7 receptor α (IL7Rα) in a dose-escalating study. RESULTS. Between 90% and 100% IL-7R occupancy and near-complete inhibition of pSTAT5 was observed at doses of RN168 1 mg/kg every other week (Q2wk) and greater. There was a significant decline in CD4+ and CD8+ effector and central memory T cells and CD4+ naive cells, but there were fewer effects on CD8+ naive T cells. The ratios of Tregs to CD4+ or CD8+ effector and central memory T cells versus baseline were increased. RNA sequencing analysis showed downmodulation of genes associated with activation, survival, and differentiation of T cells. Expression of the antiapoptotic protein Bcl-2 was reduced. The majority of treatment-emergent adverse events (TEAEs) were mild and not treatment related. Four subjects became anti–EBV IgG+ after RN168, and 2 had symptoms of active infection. The immunologic response to tetanus toxoid was preserved at doses of 1 and 3 mg/kg Q2wk but reduced at higher doses. CONCLUSIONS. This trial shows that, at dosages of 1–3 mg/kg, RN168 selectively inhibits the survival and activity of memory T cells while preserving naive T cells and Tregs. These immunologic effects may serve to eliminate pathologic T cells in autoimmune diseases. TRIAL REGISTRATION. NCT02038764. FUNDING. Pfizer Inc.
Kevan C. Herold, Samantha L. Bucktrout, Xiao Wang, Bruce W. Bode, Stephen E. Gitelman, Peter A. Gottlieb, Jing Hughes, Tenshang Joh, Janet B. McGill, Jeremy H. Pettus, Shobha Potluri, Desmond Schatz, Megan Shannon, Chandrasekhar Udata, Gilbert Wong, Matteo Levisetti, Bishu J. Ganguly, Pamela D. Garzone, the RN168 Working Group
Distinct subsets of Tregs reside in nonlymphoid tissues where they mediate unique functions. To interrogate the biology of tissue Tregs in human health and disease, we phenotypically and functionally compared healthy skin Tregs with those in peripheral blood, inflamed psoriatic skin, and metastatic melanoma. The mitochondrial enzyme, arginase 2 (ARG2), was preferentially expressed in Tregs in healthy skin, increased in Tregs in metastatic melanoma, and reduced in Tregs from psoriatic skin. ARG2 enhanced Treg suppressive capacity in vitro and conferred a selective advantage for accumulation in inflamed tissues in vivo. CRISPR-mediated deletion of this gene in primary human Tregs was sufficient to skew away from a tissue Treg transcriptional signature. Notably, the inhibition of ARG2 increased mTOR signaling, whereas the overexpression of this enzyme suppressed it. Taken together, our results suggest that Tregs express ARG2 in human tissues to both regulate inflammation and enhance their metabolic fitness.
Margaret M. Lowe, Ian Boothby, Sean Clancy, Richard S. Ahn, Wilson Liao, David N. Nguyen, Kathrin Schumann, Alexander Marson, Kelly M. Mahuron, Gillian A. Kingsbury, Zheng Liu, Priscila Munoz Sandoval, Robert Sanchez Rodriguez, Mariela L. Pauli, Keyon Taravati, Sarah T. Arron, Isaac M. Neuhaus, Hobart W. Harris, Esther A. Kim, Uk Sok Shin, Matthew F. Krummel, Adil Daud, Tiffany C. Scharschmidt, Michael D. Rosenblum
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