Tumor-infiltrating B cells (TIL-B) in breast cancer (BC) have previously been associated with improved clinical outcomes; however, their roles in tumor immunity are not currently well known. This study confirms and extends the correlation between higher TIL-B densities and positive outcomes through an analysis of HER2+ and triple-negative BC patients from the BIG 02-98 clinical trial (10-year median follow-up). Fresh tissue analyses identify an increase in TIL-B density in untreated primary BC compared with normal breast tissues, which is associated with global, CD4+, and CD8+ tumor infiltrating lymphocytes (TIL); higher tumor grades; higher proliferation; and hormone receptor negativity. All B cell differentiation stages are detectable, but significant increases in memory TIL-B are consistently present. BC with higher infiltrates are specifically characterized by germinal center TIL-B, which in turn are correlated with T follicular helper (TFH) TIL and antibody-secreting TIL-B principally located in tertiary lymphoid structures. Some TIL-B also interact directly with tumor cells. Functional analyses reveal that TIL-B are responsive to B cell receptor (BCR) stimulation ex vivo, express activation markers, and produce cytokines and Igs despite reduced expression of the antigen-presenting molecules HLA-DR and CD40. Overall, these data support the concept that ongoing humoral immune responses are generated by TIL-B and help to promote effective antitumor immunity at the tumor site.
Soizic Garaud, Laurence Buisseret, Cinzia Solinas, Chunyan Gu-Trantien, Alexandre de Wind, Gert Van den Eynden, Celine Naveaux, Jean-Nicolas Lodewyckx, Anaïs Boisson, Hughes Duvillier, Ligia Craciun, Lieveke Ameye, Isabelle Veys, Marianne Paesmans, Denis Larsimont, Martine Piccart-Gebhart, Karen Willard-Gallo
Chagas disease is a lifelong pathology resulting from Trypanosoma cruzi infection. It represents one of the most frequent causes of heart failure and sudden death in Latin America. Herein, we provide evidence that aerobic glycolytic pathway activation in monocytes drives nitric oxide (NO) production, triggering tyrosine nitration (TN) on CD8+ T cells and dysfunction in patients with chronic Chagas disease. Monocytes from patients exhibited a higher frequency of hypoxia-inducible factor 1α and increased expression of its target genes/proteins. Nonclassical monocytes are expanded in patients’ peripheral blood and represent an important source of NO. Monocytes entail CD8+ T cell surface nitration because both the frequency of nonclassical monocytes and that of NO-producing monocytes positively correlated with the percentage of TN+ lymphocytes. Inhibition of glycolysis in in vitro–infected peripheral blood mononuclear cells decreased the inflammatory properties of monocytes/macrophages, diminishing the frequency of IL-1β– and NO-producing cells. In agreement, glycolysis inhibition reduced the percentage of TN+CD8+ T cells, improving their functionality. Altogether, these results clearly show that glycolysis governs oxidative stress on monocytes and modulates monocyte–T cell interplay in human chronic Chagas disease. Understanding the pathological immune mechanisms that sustain an inflammatory environment in human pathology is key to designing improved therapies.
Liliana María Sanmarco, Natalia Eberhardt, Gastón Bergero, Luz Piedad Quebrada Palacio, Pamela Martino Adami, Laura Marina Visconti, Ángel Ramón Minguez, Yolanda Hernández-Vasquez, Eugenio Antonio Carrera Silva, Laura Morelli, Miriam Postan, Maria Pilar Aoki
Autoimmune diseases resulting from MHC class II–restricted autoantigen-specific T cell immunity include the systemic inflammatory autoimmune conditions rheumatoid arthritis and vasculitis. While currently treated with broad-acting immunosuppressive drugs, a preferable strategy is to regulate antigen-specific effector T cells (Teffs) to restore tolerance by exploiting DC antigen presentation. We targeted draining lymph node (dLN) phagocytic DCs using liposomes encapsulating 1α,25-dihydroxyvitamin D3 (calcitriol) and antigenic peptide to elucidate mechanisms of tolerance used by DCs and responding T cells under resting and immunized conditions. PD-L1 expression was upregulated in dLNs of immunized relative to naive mice. Subcutaneous administration of liposomes encapsulating OVA323–339 and calcitriol targeted dLN PD-L1hi DCs of immunized mice and reduced their MHC class II expression. OVA323–339/calcitriol liposomes suppressed expansion, differentiation, and function of Teffs and induced Foxp3+ and IL-10+ peripheral Tregs in an antigen-specific manner, which was dependent on PD-L1. Peptide/calcitriol liposomes modulated CD40 expression by human DCs and promoted Treg induction in vitro. Liposomes encapsulating calcitriol and disease-associated peptides suppressed the severity of rheumatoid arthritis and Goodpasture’s vasculitis models with suppression of antigen-specific memory T cell differentiation and function. Accordingly, peptide/calcitriol liposomes leverage DC PD-L1 for antigen-specific T cell regulation and induce antigen-specific tolerance in inflammatory autoimmune diseases.
Ryan Galea, Hendrik J. Nel, Meghna Talekar, Xiao Liu, Joshua D. Ooi, Megan Huynh, Sara Hadjigol, Kate J. Robson, Yi Tian Ting, Suzanne Cole, Karyn Cochlin, Shannon Hitchcock, Bijun Zeng, Suman Yekollu, Martine Boks, Natalie Goh, Helen Roberts, Jamie Rossjohn, Hugh H. Reid, Ben J. Boyd, Ravi Malaviya, David J. Shealy, Daniel G. Baker, Loui Madakamutil, A. Richard Kitching, Brendan J. O’Sullivan, Ranjeny Thomas
Osteoarthritis (OA) is the leading cause of joint failure, yet the underlying mechanisms remain elusive, and no approved therapies that slow progression exist. Dysregulated integrin function was previously implicated in OA pathogenesis. However, the roles of integrin αVβ3 and the integrin-associated receptor CD47 in OA remain largely unknown. Here, transcriptomic and proteomic analyses of human and murine osteoarthritic tissues revealed dysregulated expression of αVβ3, CD47, and their ligands. Using genetically deficient mice and pharmacologic inhibitors, we showed that αVβ3, CD47, and the downstream signaling molecules Fyn and FAK are crucial to OA pathogenesis. MicroPET/CT imaging of a mouse model showed elevated ligand-binding capacities of integrin αVβ3 and CD47 in osteoarthritic joints. Further, our in vitro studies demonstrated that chondrocyte breakdown products, derived from articular cartilage of individuals with OA, induced αVβ3/CD47-dependent expression of inflammatory and degradative mediators, and revealed the downstream signaling network. Our findings identify a central role for dysregulated αVβ3 and CD47 signaling in OA pathogenesis and suggest that activation of αVβ3 and CD47 signaling in many articular cell types contributes to inflammation and joint destruction in OA. Thus, the data presented here provide a rationale for targeting αVβ3, CD47, and their signaling pathways as a disease-modifying therapy.
Qian Wang, Kazuhiro Onuma, Changhao Liu, Heidi Wong, Michelle S. Bloom, Eileen E. Elliott, Richard R.L. Cao, Nick Hu, Nithya Lingampalli, Orr Sharpe, Xiaoyan Zhao, Dong Hyun Sohn, Christin M. Lepus, Jeremy Sokolove, Rong Mao, Cecilia T. Cisar, Harini Raghu, Constance R. Chu, Nicholas J. Giori, Stephen B. Willingham, Susan S. Prohaska, Zhen Cheng, Irving L. Weissman, William H. Robinson
With multifactorial etiologies, combined with disease heterogeneity and a lack of suitable diagnostic markers and therapy, endometriosis remains a major reproductive health challenge. Extracellular vesicles (EVs) have emerged as major contributors of disease progression in several conditions, including a variety of cancers; however, their role in endometriosis pathophysiology has remained elusive. Using next-generation sequencing of EVs obtained from endometriosis patient tissues and plasma samples compared with controls, we have documented that patient EVs carry unique signatures of miRNAs and long noncoding RNAs (lncRNAs) reflecting their contribution to disease pathophysiology. Mass spectrophotometry–based proteomic analysis of EVs from patient plasma and peritoneal fluid further revealed enrichment of specific pathways, as well as altered immune and metabolic processes. Functional studies in endometriotic epithelial and endothelial cell lines using EVs from patient plasma and controls clearly indicate autocrine uptake and paracrine cell proliferative roles, suggestive of their involvement in endometriosis. Multiplex cytokine analysis of cell supernatants in response to patient and control plasma–derived EVs indicate robust signatures of important inflammatory and angiogenic cytokines known to be involved in disease progression. Collectively, these findings suggest that endometriosis-associated EVs carry unique cargo and contribute to disease pathophysiology by influencing inflammation, angiogenesis, and proliferation within the endometriotic lesion microenvironment.
Kasra Khalaj, Jessica E. Miller, Harshavardhan Lingegowda, Asgerally T. Fazleabas, Steven L. Young, Bruce A. Lessey, Madhuri Koti, Chandrakant Tayade
Recently we demonstrated that ablation of the DNA methyltransferase enzyme, Dnmt3b, resulted in catabolism and progression of osteoarthritis (OA) in murine articular cartilage through a mechanism involving increased mitochondrial respiration. In this study, we identify 4-aminobutyrate aminotransferase (Abat) as a downstream target of Dnmt3b. Abat is an enzyme that metabolizes γ-aminobutyric acid to succinate, a key intermediate in the tricarboxylic acid cycle. We show that Dnmt3b binds to the Abat promoter, increases methylation of a conserved CpG sequence just upstream of the transcriptional start site, and inhibits Abat expression. Dnmt3b deletion in articular chondrocytes results in reduced methylation of the CpG sequence in the Abat promoter, which subsequently increases expression of Abat. Increased Abat expression in chondrocytes leads to enhanced mitochondrial respiration and elevated expression of catabolic genes. Overexpression of Abat in murine knee joints via lentiviral injection results in accelerated cartilage degradation following surgical induction of OA. In contrast, lentiviral-based knockdown of Abat attenuates the expression of IL-1β–induced catabolic genes in primary murine articular chondrocytes in vitro and also protects against murine articular cartilage degradation in vivo. Strikingly, treatment with the FDA-approved small-molecule Abat inhibitor, vigabatrin, significantly prevents the development of injury-induced OA in mice. In summary, these studies establish Abat as an important new target for therapies to prevent OA.
Jie Shen, Cuicui Wang, Jun Ying, Taotao Xu, Audrey McAlinden, Regis J. O’Keefe
The control of voluntary skeletal muscle contraction relies on action potentials, which send signals from the motor neuron through the neuromuscular junction (NMJ). Although dysfunction of the NMJ causes various neuromuscular diseases, a reliable in vitro system for disease modeling is currently unavailable. Here, we present a potentially novel 2-step, self-organizing approach for generating in vitro human NMJs from human induced pluripotent stem cells. Our simple and robust approach results in a complex NMJ structure that includes functional connectivity, recapitulating in vivo synapse formation. We used these in vitro NMJs to model the pathological features of spinal muscular atrophy, revealing the developmental and functional defects of NMJ formation and NMJ-dependent muscular contraction. Our differentiation system is therefore useful for investigating and understanding the physiology and pathology of human NMJs.
Chuang-Yu Lin, Michiko Yoshida, Li-Tzu Li, Akihiro Ikenaka, Shiori Oshima, Kazuhiro Nakagawa, Hidetoshi Sakurai, Eriko Matsui, Tatsutoshi Nakahata, Megumu K. Saito
The roles of macrophages in orchestrating innate immunity through phagocytosis and T lymphocyte activation have been extensively investigated. Much less understood is the unexpected role of macrophages in direct tumor regression. Tumoricidal macrophages can indeed manifest cancer immunoediting activity in the absence of adaptive immunity. We investigated direct macrophage cytotoxicity in malignant pleural mesothelioma, a lethal cancer that develops from mesothelial cells of the pleural cavity after occupational asbestos exposure. In particular, we analyzed the cytotoxic activity of mouse RAW264.7 macrophages upon cell-cell contact with autologous AB1/AB12 mesothelioma cells. We show that macrophages killed mesothelioma cells by oxeiptosis via a mechanism involving enhancer of zeste homolog 2 (EZH2), a histone H3 lysine 27–specific (H3K27-specific) methyltransferase of the polycomb repressive complex 2 (PRC2). A selective inhibitor of EZH2 indeed impaired RAW264.7-directed cytotoxicity and concomitantly stimulated the PD-1 immune checkpoint. In the immunocompetent BALB/c model, RAW264.7 macrophages pretreated with the EZH2 inhibitor failed to control tumor growth of AB1 and AB12 mesothelioma cells. Blockade of PD-1 engagement restored macrophage-dependent antitumor activity. We conclude that macrophages can be directly cytotoxic for mesothelioma cells independent of phagocytosis. Inhibition of the PRC2 EZH2 methyltransferase reduces this activity because of PD-1 overexpression. Combination of PD-1 blockade and EZH2 inhibition restores macrophage cytotoxicity.
Malik Hamaidia, Hélène Gazon, Clotilde Hoyos, Gabriela Brunsting Hoffmann, Renaud Louis, Bernard Duysinx, Luc Willems
Hereditary renal cystic diseases are characterized by defects in primary cilia of renal tubular epithelial cells and abnormality of tubular epithelium, which ultimately result in the development of renal cysts. However, the mechanism leading from abnormality of the tubular epithelium to cystogenesis is not well understood. In this report, we demonstrate a critical role for Robo2 in regulating epithelial development, including ciliogenesis, polarization, and differentiation. We found that Robo2 deficiency results in cystic kidneys, and the cyst cells showed defective cilia and polarity defects in tubular epithelium. The cyst cells, less than terminally differentiated, continue to proliferate. We further established that Robo2 works with p53 as well as polarity and ciliary proteins (Par3, PKCς, ZO-2, and Claudin-2) to regulate these processes. Robo2 binds to Baiap2 (also known as IRSp53) through the IRSp53/MIM homology domain in renal epithelial cells. This binding allows Robo2 to phosphorylate MDM2 at Ser166 via Baiap2 and maintain p53 homeostasis. Disruption of the Robo2-Baiap2 complex causes MDM2 to be subjected to dephosphorylation, leading to a high level of active p53, and initiated p53-mediated cellular senescence via p21 and decreased the expression of ZO-1, ZO-2, PKCς, Par3, and Claudin-2 proteins, resulting in defects in epithelial development, including ciliogenesis, polarization, and differentiation. Importantly, double knockout of Robo2 and p53 rescued all the epithelial defects in kidneys compared with those in Robo2-knockout kidneys. Taken together, the present results demonstrate that Robo2 deficiency causes renal cystic disease, which is largely dependent on defective Robo2-Baiap2 integrated signaling in kidneys.
Qinggang Li, Shaoyuan Cui, Qian Ma, Ying Liu, Hongyu Yu, GuangRui Geng, Ewud Agborbesong, Chongyu Ren, Kai Wei, Yingjie Zhang, Jurong Yang, Xueyuan Bai, Guangyan Cai, Yuansheng Xie, Xiaogang Li, Xiangmei Chen
The cellular origins of glomerulosclerosis involve activation of parietal epithelial cells (PECs) and progressive podocyte depletion. While mammalian target of rapamycin–mediated (mTOR-mediated) podocyte hypertrophy is recognized as an important signaling pathway in the context of glomerular disease, the role of podocyte hypertrophy as a compensatory mechanism preventing PEC activation and glomerulosclerosis remains poorly understood. In this study, we show that glomerular mTOR and PEC activation–related genes were both upregulated and intercorrelated in biopsies from patients with focal segmental glomerulosclerosis (FSGS) and diabetic nephropathy, suggesting both compensatory and pathological roles. Advanced morphometric analyses in murine and human tissues identified podocyte hypertrophy as a compensatory mechanism aiming to regulate glomerular functional integrity in response to somatic growth, podocyte depletion, and even glomerulosclerosis — all of this in the absence of detectable podocyte regeneration. In mice, pharmacological inhibition of mTOR signaling during acute podocyte loss impaired hypertrophy of remaining podocytes, resulting in unexpected albuminuria, PEC activation, and glomerulosclerosis. Exacerbated and persistent podocyte hypertrophy enabled a vicious cycle of podocyte loss and PEC activation, suggesting a limit to its beneficial effects. In summary, our data highlight a critical protective role of mTOR-mediated podocyte hypertrophy following podocyte loss in order to preserve glomerular integrity, preventing PEC activation and glomerulosclerosis.
Victor G. Puelles, James W. van der Wolde, Nicola Wanner, Markus W. Scheppach, Luise A. Cullen-McEwen, Tillmann Bork, Maja T. Lindenmeyer, Lukas Gernhold, Milagros N. Wong, Fabian Braun, Clemens D. Cohen, Michelle M. Kett, Christoph Kuppe, Rafael Kramann, Turgay Saritas, Claudia R. van Roeyen, Marcus J. Moeller, Leon Tribolet, Richard Rebello, Yu B.Y. Sun, Jinhua Li, Gerard Müller-Newen, Michael D. Hughson, Wendy E. Hoy, Fermin Person, Thorsten Wiech, Sharon D. Ricardo, Peter G. Kerr, Kate M. Denton, Luc Furic, Tobias B. Huber, David J. Nikolic-Paterson, John F. Bertram
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