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
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
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
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
Chronic graft-versus-host disease (cGvHD) remains a major complication of allogeneic stem cell transplantation requiring novel therapies. CD146 and CCR5 are expressed by activated T cells and associated with increased T cell migration capacity and Th17 polarization. We performed a multiparametric flow cytometry analysis in a cohort of 40 HSCT patients together with a cGvHD murine model to understand the role of CD146-expressing subsets. We observed an increased frequency of CD146+ CD4 T cells in the 20 patients with active cGvHD with enhanced RORγt expression. This Th17-prone subset was enriched for cells coexpressing CD146 and CCR5 that harbor mixed Th1/Th17 features and were more frequent in cGvHD patients. Utilizing a murine cGvHD model with bronchiolitis obliterans (BO), we observed that donor T cells from CD146-deficient mice versus those from WT mice caused significantly reduced pulmonary cGvHD. Reduced cGvHD was not the result of failed germinal center B cell or T follicular helper cell generation. Instead, CD146-deficient T cells had significantly lower pulmonary macrophage infiltration and T cell CCR5, IL-17, and IFN-γ coexpression, suggesting defective pulmonary end-organ effector mechanisms. We, thus, evaluated the effect of TMP778, a small-molecule RORγt activity inhibitor. TMP778 markedly alleviated cGvHD in murine models similarly to agents targeting the Th17 pathway, such as STAT3 inhibitor or IL-17–blocking antibody. Our data suggest CD146-expressing T cells as a cGvHD biomarker and suggest that targeting the Th17 pathway may represent a promising therapy for cGvHD.
Edouard Forcade, Katelyn Paz, Ryan Flynn, Brad Griesenauer, Tohti Amet, Wei Li, Liangyi Liu, Giorgos Bakoyannis, Di Jiang, Hong Wei Chu, Mercedes Lobera, Jianfei Yang, David S. Wilkes, Jing Du, Kate Gartlan, Geoffrey R. Hill, Kelli P.A. MacDonald, Eduardo L. Espada, Patrick Blanco, Jonathan S. Serody, John Koreth, Corey S. Cutler, Joseph H. Antin, Robert J. Soiffer, Jerome Ritz, Sophie Paczesny, Bruce R. Blazar
The tumor microenvironment imposes physical and functional constraints on the antitumor efficacy of adoptive T cell immunotherapy. Preclinical testing of different T cell preparations can help in the selection of efficient immune therapies, but in vivo models are expensive and cumbersome to develop, while classical in vitro 2D models cannot recapitulate the spatiotemporal dynamics experienced by T cells targeting cancer. Here, we describe an easily customizable 3D model, in which the tumor microenvironment conditions are modulated and the functionality of different T cell preparations is tested. We incorporate human cancer hepatocytes as a single cell or as tumor cell aggregates in a 3D collagen gel region of a microfluidic device. Human T cells engineered to express tumor-specific T cell receptors (TCR–T cells) are then added in adjacent channels. The TCR–T cells’ ability to migrate and kill the tumor target and the profile of soluble factors were investigated under conditions of varying oxygen levels and in the presence of inflammatory cytokines. We show that only the 3D model detects the effect that oxygen levels and the inflammatory environment impose on engineered TCR–T cell function, and we also used the 3D microdevice to analyze the TCR–T cell efficacy in an immunosuppressive scenario. Hence, we show that our microdevice platform enables us to decipher the factors that can alter T cell function in 3D and can serve as a preclinical assay to tailor the most efficient immunotherapy configuration for a specific therapeutic goal.
Andrea Pavesi, Anthony T. Tan, Sarene Koh, Adeline Chia, Marta Colombo, Emanuele Antonecchia, Carlo Miccolis, Erica Ceccarello, Giulia Adriani, Manuela T. Raimondi, Roger D. Kamm, Antonio Bertoletti
Zika virus (ZIKV) is an emerging mosquito-transmitted flavivirus that shares a considerable degree of homology with dengue virus (DENV). Here, we examined longitudinal antibody response against ZIKV during natural infection in 2 convalescent individuals. By decomposing the antibody recognition into DI/DII and DIII of the E glycoprotein, we showed their development in humans followed a spatiotemporal hierarchy. Plasma binding to DI/DII appeared to peak and wane during early infection with extensive cross-reactivity with DI/DII of DENV. Binding to DIII, however, peaked early but persisted months into the infection without detectable cross-reactivity with DIII of DENV. A clear trend of increase in DIII-specific neutralizing activity was observed over the course of infection. mAbs isolated during early infection are largely DI/DII specific, weakly neutralizing, and highly cross-reactive with DENV, while those from later infection are more diverse in recognition, potently neutralizing, and ZIKV specific. The most potent neutralizing mAb targeting the DIII provided 100% protection in mice from lethal ZIKV infection and could therefore serve as a promising candidate for antibody-based therapy and prevention. The dynamic features unveiled here will assist us to better understand the pathogenesis of ZIKV infection and inform rational design of vaccines.
Lei Yu, Ruoke Wang, Fei Gao, Min Li, Jianying Liu, Jian Wang, Wenxin Hong, Lingzhai Zhao, Yingfen Wen, Chibiao Yin, Hua Wang, Qi Zhang, Yangyang Li, Panpan Zhou, Rudian Zhang, Yang Liu, Xiaoping Tang, Yongjun Guan, Cheng-Feng Qin, Ling Chen, Xuanling Shi, Xia Jin, Gong Cheng, Fuchun Zhang, Linqi Zhang
Rituximab is a therapeutic anti-CD20 monoclonal antibody widely used to treat B cell lymphoma and autoimmune diseases, such as rheumatic arthritis, systemic lupus erythematosus, and autoimmune blistering skin diseases (AIBD). While rituximab fully depletes peripheral blood B cells, it remains unclear whether some preexisting B cell memory to pathogens or vaccines may survive depletion, especially in lymphoid tissues, and if these memory B cells can undergo homeostatic expansion during recovery from depletion. The limited data available on vaccine efficacy in this setting have been derived from rituximab-treated patients receiving concomitant chemotherapy or other potent immunosuppressants. Here, we present an in-depth analysis of seasonal influenza vaccine responses in AIBD patients previously treated with rituximab, who generally did not receive additional therapeutic interventions. We found that, despite a lack of influenza-specific memory B cells in the blood, patients mount robust recall responses to vaccination, comparable to healthy controls, both at a cellular and a serological level. Repertoire analyses of plasmablast responses suggest that they likely derive from a diverse pool of tissue-resident memory cells, refractory to depletion. Overall, these data have important implications for establishing an effective vaccine schedule for AIBD patients and the clinical care of rituximab-treated patients in general and contribute to our basic understanding of maintenance of normal and pathogenic human B cell memory.
Alice Cho, Bridget Bradley, Robert Kauffman, Lalita Priyamvada, Yevgeniy Kovalenkov, Ron Feldman, Jens Wrammert
Cancer cells can inhibit effector T cells (Teff) through both immunomodulatory receptors and the impact of cancer metabolism on the tumor microenvironment. Indeed, Teff require high rates of glucose metabolism, and consumption of essential nutrients or generation of waste products by tumor cells may impede essential T cell metabolic pathways. Clear cell renal cell carcinoma (ccRCC) is characterized by loss of the tumor suppressor von Hippel-Lindau (VHL) and altered cancer cell metabolism. Here, we assessed how ccRCC influences the metabolism and activation of primary patient ccRCC tumor infiltrating lymphocytes (TIL). CD8 TIL were abundant in ccRCC, but they were phenotypically distinct and both functionally and metabolically impaired. ccRCC CD8 TIL were unable to efficiently uptake glucose or perform glycolysis and had small, fragmented mitochondria that were hyperpolarized and generated large amounts of ROS. Elevated ROS was associated with downregulated mitochondrial SOD2. CD8 T cells with hyperpolarized mitochondria were also visible in the blood of ccRCC patients. Importantly, provision of pyruvate to bypass glycolytic defects or scavengers to neutralize mitochondrial ROS could partially restore TIL activation. Thus, strategies to improve metabolic function of ccRCC CD8 TIL may promote the immune response to ccRCC.
Peter J. Siska, Kathryn E. Beckermann, Frank M. Mason, Gabriela Andrejeva, Allison R. Greenplate, Adam B. Sendor, Yun-Chen J. Chiang, Armando L. Corona, Lelisa F. Gemta, Benjamin G. Vincent, Richard C. Wang, Bumki Kim, Jiyong Hong, Chiu-lan Chen, Timothy N. Bullock, Jonathan M. Irish, W. Kimryn Rathmell, Jeffrey C. Rathmell
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