Evaluation of T lymphocyte frequency provides prognostic information for patients with oral squamous cell cancer (OSCC). However, the effect of simultaneously evaluating T cell frequency and assessing suppressive elements and defects in antigen-processing machinery (APM) has not been clarified. Simultaneous characterization of CD3+, CD8+, FoxP3+, CD163+, and PD-L1+ cells using multispectral imaging was performed on sections from 119 patients with HPV– OSCC. Expression of β2-microglobulin, MHC class I heavy chain, and large multifunctional peptidase 10 was quantified, and all data were correlated with patient outcome. We found that, consistent with previous reports, high numbers of CD8+ T cells at the invasive margin correlated significantly with prolonged overall survival (OS), while the number of FoxP3+ or PD-L1+ cells did not. Compiling the number of FoxP3+ or PD-L1+ cells within 30 μm of CD8+ T cells identified a significant association with a high number of suppressive elements close to CD8+ T cells and reduced OS. Integrating this information into a cumulative suppression index (CSI) increased correlation with OS. Incorporating tumor expression levels of APM components with CSI further improved prognostic power. This multiparametric immune profiling may be useful for stratifying patients with OSCC for clinical trials.
Zipei Feng, Daniel Bethmann, Matthias Kappler, Carmen Ballesteros-Merino, Alexander Eckert, R. Bryan Bell, Allen Cheng, Tuan Bui, Rom Leidner, Walter J. Urba, Kent Johnson, Clifford Hoyt, Carlo B. Bifulco, Juergen Bukur, Claudia Wickenhauser, Barbara Seliger, Bernard A. Fox
Graft-versus-host disease (GVHD) induces pathological damage in peripheral target organs leading to well-characterized, organ-specific clinical manifestations. Patients with GVHD, however, can also have behavioral alterations that affect overall cognitive function, but the extent to which GVHD alters inflammatory and biochemical pathways in the brain remain poorly understood. In the current study, we employed complementary murine GVHD models to demonstrate that alloreactive donor T cells accumulate in the brain and affect a proinflammatory cytokine milieu that is associated with specific behavioral abnormalities. Host IL-6 was identified as a pivotal cytokine mediator, as was host indoleamine 2,3-dioxygenase (IDO-1), which was upregulated in GVHD in an IL-6–dependent manner in microglial cells and was accompanied by dysregulated tryptophan metabolism in the dorsal raphe nucleus and prefrontal cortex. Blockade of the IL-6 signaling pathway significantly reduced donor T cell accumulation, inflammatory cytokine gene expression, and host microglial cell expansion, but did not reverse GVHD-induced tryptophan metabolite dysregulation. Thus, these results indicate that inhibition of IL-6 signaling attenuates neuroinflammation, but does not reverse all of the metabolic abnormalities in the brain during GVHD, which may also have implications for the treatment of neurotoxicity occurring after other T cell–based immune therapies with IL-6–directed approaches.
Ludovic Belle, Vivian Zhou, Kara L. Stuhr, Margaret Beatka, Emily M. Siebers, Jennifer M. Knight, Michael W. Lawlor, Casey Weaver, Misato Hashizume, Cecilia J. Hillard, William R. Drobyski
Dengue virus (DENV) is the most prevalent mosquito-borne virus causing human disease. Of the 4 DENV serotypes, epidemiological data suggest that DENV-2 secondary infections are associated with more severe disease than DENV-4 infections. Mass cytometry by time-of-flight (CyTOF) was used to dissect immune changes induced by DENV-2 and DENV-4 in human DCs, the initial targets of primary infections that likely affect infection outcomes. Strikingly, DENV-4 replication peaked earlier and promoted stronger innate immune responses, with increased expression of DC activation and migration markers and increased cytokine production, compared with DENV-2. In addition, infected DCs produced higher levels of inflammatory cytokines compared with bystander DCs, which mainly produced IFN-induced cytokines. These high-dimensional analyses during DENV-2 and DENV-4 infections revealed distinct viral signatures marked by different replication strategies and antiviral innate immune induction in DCs, which may result in different viral fitness, transmission, and pathogenesis.
Rebecca E. Hamlin, Adeeb Rahman, Theodore R. Pak, Kevin Maringer, Ignacio Mena, Dabeiba Bernal-Rubio, Uma Potla, Ana M. Maestre, Anthony C. Fredericks, El-ad D. Amir, Andrew Kasarskis, Irene Ramos, Miriam Merad, Ana Fernandez-Sesma
Sepsis can induce an overwhelming systemic inflammatory response, resulting in organ damage and death. Suppressor of cytokine signaling 1 (SOCS1) negatively regulates signaling by cytokine receptors and Toll-like receptors (TLRs). However, the cellular targets and molecular mechanisms for SOCS1 activity during polymicrobial sepsis are unknown. To address this, we utilized a cecal ligation and puncture (CLP) model for sepsis; C57BL/6 mice subjected to CLP were then treated with a peptide (iKIR) that binds the SOCS1 kinase inhibitory region (KIR) and blocks its activity. Treatment with iKIR increased CLP-induced mortality, bacterial burden, and inflammatory cytokine production. Myeloid cell–specific SOCS1 deletion (Socs1Δmyel) mice were also more susceptible to sepsis, demonstrating increased mortality, higher bacterial loads, and elevated inflammatory cytokines, compared with Socs1fl littermate controls. These effects were accompanied by macrophage metabolic reprograming, as evidenced by increased lactic acid production and elevated expression of the glycolytic enzymes hexokinase, lactate dehydrogenase A, and glucose transporter 1 in septic Socs1Δmyel mice. Upregulation was dependent on the STAT3/HIF-1α/glycolysis axis, and blocking glycolysis ameliorated increased susceptibility to sepsis in iKIR-treated CLP mice. These results reveal a role of SOCS1 as a regulator of metabolic reprograming that prevents overwhelming inflammatory response and organ damage during sepsis.
Annie Rocio Piñeros Alvarez, Nicole Glosson-Byers, Stephanie Brandt, Soujuan Wang, Hector Wong, Sarah Sturgeon, Brian Paul McCarthy, Paul R. Territo, Jose Carlos Alves-Filho, C. Henrique Serezani
BACKGROUND. Both seasonal and novel avian influenza viruses can result in severe infections requiring hospitalization. Anti-influenza antibodies (Abs) with Fc-mediated effector functions, such as Ab-dependent cellular cytotoxicity (ADCC), are of growing interest in control of influenza but have not previously been studied during severe human infections. As such, the objective of this study was to examine Fc-mediated Ab functions in humans hospitalized with influenza infection. METHODS. Serum Ab response was studied in subjects hospitalized with either pandemic H7N9 avian influenza virus in China (n = 18) or circulating seasonal influenza viruses in Melbourne, Australia (n = 16). Recombinant soluble Fc receptor dimer ELISAs, natural killer (NK) cell activation assays, and Ab-dependent killing assays with influenza-infected target cells were used to assess the Fc functionality of anti-influenza hemagglutinin (HA) Abs during severe human influenza infection. RESULTS. We found that the peak generation of Fc functional HA Abs preceded that of neutralizing Abs for both severe H7N9 and seasonal influenza infections. Subjects who succumbed to complications of H7N9 infection demonstrated reduced HA-specific Fc receptor–binding Abs (in magnitude and breadth) immediately prior to death compared with those who survived. Subjects who recovered from H7N9 and severe seasonal influenza infections demonstrated increased Fc receptor–binding Abs not only against the homologous infecting strain but against HAs from different influenza A subtypes. CONCLUSION. Collectively, survivors of severe influenza infection rapidly generate a functional Ab response capable of mediating ADCC against divergent influenza viruses. Broadly binding HA Abs with Fc-mediated functions may be a useful component of protective immunity to severe influenza infection. FUNDING. The National Health and Medical Research Council ([NHMRC] grants 1023294, 1041832, and 1071916), the Australian Department of Health, and the joint University of Melbourne/Fudan University International Research and Research Training Fund provided funding for this study.
Hillary A. Vanderven, Lu Liu, Fernanda Ana-Sosa-Batiz, Thi H.O. Nguyen, Yanmin Wan, Bruce Wines, P. Mark Hogarth, Danielle Tilmanis, Arnold Reynaldi, Matthew S. Parsons, Aeron C. Hurt, Miles P. Davenport, Tom Kotsimbos, Allen C. Cheng, Katherine Kedzierska, Xiaoyan Zhang, Jianqing Xu, Stephen J. Kent
A central issue for adoptive cellular immunotherapy is overcoming immunosuppressive signals to achieve tumor clearance. While γδ T cells are known to be potent cytolytic effectors that can kill a variety of cancers, it is not clear whether they are inhibited by suppressive ligands expressed in tumor microenvironments. Here, we have used a powerful preclinical model where EBV infection drives the de novo generation of human B cell lymphomas in vivo, and autologous T lymphocytes are held in check by PD-1/CTLA-4–mediated inhibition. We show that a single dose of adoptively transferred Vδ2+ T cells has potent antitumor effects, even in the absence of checkpoint blockade or activating compounds. Vδ2+ T cell immunotherapy given within the first 5 days of EBV infection almost completely prevented the outgrowth of tumors. Vδ2+ T cell immunotherapy given more than 3 weeks after infection (after neoplastic transformation is evident) resulted in a dramatic reduction in tumor burden. The immunotherapeutic Vδ2+ T cells maintained low cell surface expression of PD-1 in vivo, and their recruitment to tumors was followed by a decrease in B cells expressing PD-L1 and PD-L2 inhibitory ligands. These results suggest that adoptively transferred PD-1lo Vδ2+ T cells circumvent the tumor checkpoint environment in vivo.
Nicholas A. Zumwalde, Akshat Sharma, Xuequn Xu, Shidong Ma, Christine L. Schneider, James C. Romero-Masters, Amy W. Hudson, Annette Gendron-Fitzpatrick, Shannon C. Kenney, Jenny E. Gumperz
Today, it is known that autoimmune diseases start a long time before clinical symptoms appear. Anti-citrullinated protein antibodies (ACPAs) appear many years before the clinical onset of rheumatoid arthritis (RA). However, it is still unclear if and how ACPAs are arthritogenic. To better understand the molecular basis of pathogenicity of ACPAs, we investigated autoantibodies reactive against the C1 epitope of collagen type II (CII) and its citrullinated variants. We found that these antibodies are commonly occurring in RA. A mAb (ACC1) against citrullinated C1 was found to cross-react with several noncitrullinated epitopes on native CII, causing proteoglycan depletion of cartilage and severe arthritis in mice. Structural studies by X-ray crystallography showed that such recognition is governed by a shared structural motif “RG-TG” within all the epitopes, including electrostatic potential-controlled citrulline specificity. Overall, we have demonstrated a molecular mechanism that explains how ACPAs trigger arthritis.
Changrong Ge, Dongmei Tong, Bibo Liang, Erik Lönnblom, Nadine Schneider, Cecilia Hagert, Johan Viljanen, Burcu Ayoglu, Roma Stawikowska, Peter Nilsson, Gregg B. Fields, Thomas Skogh, Alf Kastbom, Jan Kihlberg, Harald Burkhardt, Doreen Dobritzsch, Rikard Holmdahl
Rejection affects greater than 80% of face transplants, yet no diagnostic criteria for antibody-mediated rejection (AMR) following face transplantation have been established. Given that different treatment strategies are required to address AMR and T cell–mediated rejection (TCMR), there is a critical need to delineate the features that can differentiate these two alloimmune responses. Here, we report the longitudinal immunological examination of what we believe to be the first and only highly sensitized recipient of a crossmatch-positive face transplant up to 4 years following transplantation. We conducted gene expression profiling on allograft biopsies collected during suspected AMR and TCMR episodes as well as during 5 nonrejection time points. Our data suggest that there are distinctive molecular features in AMR, characterized by overexpression of endothelial-associated genes, including ICAM1, VCAM1, and SELE. Although our findings are limited to a single patient, these findings highlight the potential importance of developing and implementing molecular markers to differentiate AMR from TCMR to guide clinical management. Furthermore, our case illustrates that molecular assessment of allograft biopsies offers the potential for new insights into the mechanisms underlying rejection. Finally, our medium-term outcomes demonstrate that face transplantation in a highly sensitized patient with a positive preoperative crossmatch is feasible and manageable.
Thet Su Win, Naoka Murakami, Thiago J. Borges, Anil Chandraker, George Murphy, Christine Lian, Victor Barrera, Shannan Ho Sui, David Schoenfeld, Jessica Teague, Ericka Bueno, Stefan G. Tullius, Bohdan Pomahac, Rachael A. Clark, Leonardo V. Riella
In recent years, the extent of our vulnerability to misinterpretation due to poorly characterized reagents has become an issue of great concern. Antibody reagents have been identified as a major source of error, contributing to the “reproducibility crisis.” In the current report, we define an additional dimension of the crisis; in particular, we define variation of the targets being analyzed. We report that natural variation in the immunoglobulin “constant” region alters the reactivity with commonly used subtype-specific anti-IgG reagents, resulting in cross-reactivity of polyclonal regents with inappropriate targets and blind spots of monoclonal reagents for desired targets. This raises the practical concern that numerous studies characterizing IgG subtypes in human disease may contain errors due to such previously unappreciated defects. These studies also focus attention on the broader concern that genetic variation may affect the performance of any laboratory or research test that uses antibodies for detection.
Heather L. Howie, Meghan Delaney, Xiaohong Wang, Lay See Er, Linda Kapp, Jenna N. Lebedev, James C. Zimring
We previously showed that Th1/type 1 inflammation marked by increased IFN-γ levels in the airways can be appreciated in 50% of patients with severe asthma, despite high dose corticosteroid (CS) treatment. We hypothesized that a downstream target of IFN-γ, CXCL10, which recruits Th1 cells via the cognate receptor CXCR3, is an important contributor to Th1high asthma and CS unresponsiveness. We show high levels of CXCL10 mRNA closely associated with IFNG levels in the BAL cells of 50% of severe asthmatics and also in the airways of mice subjected to a severe asthma model, both in the context of high-dose CS treatment. The inability of CS to dampen IFNG or CXCL10 expression was not because of impaired nuclear translocation of the glucocorticoid receptor (GR) or its transactivational functions. Rather, in the presence of CS and IFN-γ, STAT1 and GR were recruited on critical regulatory elements in the endogenous CXCL10 promoter in monocytes, albeit without any abatement of CXCL10 gene expression. High CXCL10 gene expression was also associated with a mast cell signature in both humans and mice, CXCR3 being also expressed by mast cells. These findings suggest that the IFN-γ–CXCL10 axis plays a central role in persistent type 1 inflammation that may be facilitated by CS therapy through GR-STAT1 cooperation converging on the CXCL10 promoter.
Marc Gauthier, Krishnendu Chakraborty, Timothy B. Oriss, Mahesh Raundhal, Sudipta Das, Jie Chen, Rachael Huff, Ayan Sinha, Merritt Fajt, Prabir Ray, Sally E. Wenzel, Anuradha Ray
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