Altered glycosylation of IgG4 promotes lectin complement pathway activation in anti-PLA2R1 associated membranous nephropathy

• Text
• PDF

Abstract

Primary membranous nephropathy (pMN) is a leading cause of the nephrotic syndrome in adults. In most cases, this autoimmune kidney disease is associated with autoantibodies against the M-type phospholipase A2 receptor (PLA2R1) expressed on kidney podocytes, but the mechanisms leading to glomerular damage remain elusive. Here, we developed a cell culture model using human podocytes and found that anti-PLA2R1 positive pMN patient sera or isolated IgG4, but not IgG4-depleted sera, induce proteolysis of the two essential podocyte proteins synaptopodin and NEPH1 in the presence of complement, resulting in perturbations of the podocyte cytoskeleton. Specific blockade of the lectin pathway prevented degradation of synaptopodin and NEPH1. Anti-PLA2R1-IgG4 directly bound mannose-binding lectin in a glycosylation-dependent manner. In a cohort of pMN patients, we identified increased levels of galactose-deficient IgG4, which correlated with anti-PLA2R1-titers and podocyte damage induced by patient sera. Assembly of the terminal C5b-9 complement complex and activation of the complement receptors C3aR1 or C5aR1 was required to induce proteolysis of synaptopodin and NEPH1 by two distinct proteolytic pathways, mediated by cysteine and aspartic proteinases, respectively. Together, these results demonstrate a mechanism by which aberrantly glycosylated IgG4 activates the lectin pathway and induces podocyte injury in primary membranous nephropathy.

Authors

George Haddad, Johan M. Lorenzen, Hong Ma, Noortje de Haan, Harald Seeger, Christelle Zaghrini, Simone Brandt, Malte Kölling, Urs Wegmann, Bence Kiss, Gábor Pál, Péter Gál, Rudolf P. Wuthrich, Manfred Wuhrer, Laurence H. Beck, David J. Salant, Gérard Lambeau, Andreas D. Kistler

Cyclin A2 maintains colon homeostasis and is a prognostic factor in CRC

• Text
• PDF

Abstract

To clarify the function of cyclin A2 in colon homeostasis and colorectal cancer (CRC), we generated mice deficient for cyclin A2 in colonic epithelial cells (CEC). Colons of those mice displayed architectural changes in the mucosa, and signs of inflammation as well as an increased proliferation of CEC associated with the appearance of low- and high-grade dysplasia. The main initial events triggering those alterations in cyclin A2 deficient CEC appear to be abnormal mitoses and DNA damage. Cyclin A2 deletion in CEC promoted the development of dysplasia and adenocarcinomas in the murine colitis-associated cancer model. We next explored the status of cyclin A2 expression in clinical CRC samples at the mRNA and protein level and found higher expression in tumors of stage I and II patients compared to those of stage III and IV. A meta-analysis of 11 transcriptome datasets comprising 2,239 primary CRC tumors displayed different CCNA2 (the mRNA coding for cyclin A2) expression levels among the CRC tumor subtypes with highest in CMS1 and lowest in CMS4. Moreover, high expression of CCNA2 was found to be a new independent prognosis factor for CRC tumors.

Authors

Yuchen Guo, Monica Gabola, Rossano Lattanzio, Conception Paul, Valérie Pinet, Ruizhi Tang, Hulya Turali, Julie Bremond, Ciro Longobardi, Chloé Maurizy, Quentin Da Costa, Pascal Finetti, Florence Boissière-Michot, Benjamin Rivière, Céline Lemmers, Séverine Garnier, François Bertucci, Inti Zlobec, Karim Chebli, Jamal Tazi, Rania Azar, Jean-Marie Blanchard, Peter Sicinski, Emilie Mamessier, Bénédicte Lemmers, Michael Hahne

Inhibition of 2-Hydroxyglutarate Elicits Metabolic-reprograming and Mutant IDH1 Glioma Immunity in Mice

• Text
• PDF

Abstract

Mutant isocitrate-dehydrogenase-1 (IDH1-R132H; mIDH1) is a hallmark of adult gliomas. Lower grade mIDH1 gliomas are classified into two molecular subgroups: (i) 1p/19q co-deletion/TERT-promoter mutations or (ii) inactivating mutations in α-thalassemia/mental retardation syndrome X-linked (ATRX) and TP53. This work, focuses on gliomas’ subtype harboring mIDH1, TP53 and ATRX inactivation. IDH1-R132H is a gain-of-function mutation that converts α-ketoglutarate into 2-hydroxyglutarate (D-2HG). The role of D-2HG within the tumor microenvironment of mIDH1/mATRX/mTP53 gliomas remains unexplored. Inhibition of D-2HG, when used as monotherapy or in combination with radiation and temozolomide (IR/TMZ), led to increased median survival (MS) of mIDH1 glioma bearing mice. Also, D-2HG inhibition elicited anti-mIDH1 glioma immunological memory. In response to D-2HG inhibition, PD-L1 expression levels on mIDH1-glioma cells increased to similar levels as observed in wild-type-IDH1 gliomas. Thus, we combined D-2HG inhibition/IR/TMZ with anti-PDL1 immune checkpoint-blockade and observed complete tumor regression in 60% of mIDH1 glioma bearing mice. This combination strategy reduced T-cell exhaustion and favored the generation of memory CD8+ T-cells. Our findings demonstrate that metabolic reprogramming elicits anti-mIDH1 glioma immunity, leading to increased MS and immunological memory. Our preclinical data supports the testing of IDH-R132H inhibitors in combination with IR/TMZ and anti-PDL1 as targeted therapy for mIDH1/mATRX/mTP53 glioma patients.

Authors

Padma Kadiyala, Stephen V. Carney, Jessica C. Gauss, Maria B. Garcia-Fabiani, Santiago Haase, Mahmoud S. Alghamri, Felipe J. Núñez, Yayuan Liu, Minzhi Yu, Ayman W. Taher, Fernando M. Nunez, Dan Li, Marta B. Edwards, Celina G. Kleer, Henry Appelman, Yilun Sun, Lili Zhao, James J. Moon, Anna Schwendeman, Pedro R. Lowenstein, Maria G. Castro

Regulation and targeting of androgen receptor nuclear localization in castration-resistant prostate cancer

• Text
• PDF

Abstract

Androgen receptor (AR) nuclear localization is necessary for its activation as a transcription factor. Defining the mechanisms regulating AR nuclear localization in androgen-sensitive cells, and how these mechanisms are dysregulated in castration-resistant prostate cancer (CRPC) cells are fundamentally important and clinically relevant. According to the classical model of AR intracellular trafficking, androgens induce AR nuclear import and androgen withdrawal causes AR nuclear export. The present study led to an updated model that AR could be imported in the absence of androgens, ubiquitinated, and degraded in the nucleus. Androgen withdrawal caused nuclear AR degradation but not export. In comparison to their parental androgen-sensitive LNCaP prostate cancer cells, castration-resistant C4-2 cells exhibited reduced nuclear AR polyubiquitination and increased nuclear AR level. We previously identified 3-(4-chlorophenyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazole (CPPI) in a high throughput screen for its inhibition of androgen-independent AR nuclear localization in CRPC cells. The current study showed that CPPI was a novel competitive AR antagonist capable of enhancing AR interaction with its E3 ligase MDM2, and degradation of AR in the nuclei of CRPC cells. Also, CPPI blocked androgen-independent AR nuclear import. Overall, these findings suggest the feasibility of targeting androgen-independent AR nuclear import and stabilization, two necessary steps leading to AR nuclear localization and activation in CRPC cells, with small molecule inhibitors.

Authors

Shidong Lv, Qiong Song, Guang Chen, Erdong Cheng, Wei Chen, Ryan Cole, Zeyu Wu, Laura E. Pascal, Ke Wang, Peter Wipf, Joel B. Nelson, Qiang Wei, Wenhua Huang, Zhou Wang

Active bacterial modification of the host environment through RNA Polymerase II inhibition

• Text
• PDF

Abstract

Unlike pathogens, which attack the host, commensal bacteria create a state of friendly coexistence. Here, we identified a mechanism of bacterial adaptation to the host niche, where they reside. Asymptomatic carrier strains were shown to inhibit RNA Polymerase II (Pol II) in host cells by targeting Ser2 phosphorylation; a step required for productive mRNA elongation. Assisted by a rare, spontaneous loss-of-function mutant from a human carrier, the bacterial NlpD protein was identified as a Pol II inhibitor. After internalization by host cells, NlpD was shown to target constituents of the Pol II phosphorylation complex (RPB1 and PAF1C), attenuating host gene expression. Therapeutic efficacy of the rNlpD protein was demonstrated in a urinary tract infection model, by reduced tissue pathology, accelerated bacterial clearance and attenuated Pol II-dependent gene expression. The findings suggest an intriguing, evolutionarily conserved mechanism for bacterial modulation of host gene expression, with a remarkable therapeutic potential.

Authors

Inès Ambite, Nina A. Filenko, Elisabed Zaldastanishvili, Daniel S.C. Butler, Thi Hien Tran, Arunima Chaudhuri, Parisa Esmaeili, Shahram Ahmadi, Sanchari Paul, Björn Wullt, Johannes Putze, Swaine L. Chen, Ulrich Dobrindt, Catharina Svanborg

An unbiased approach to defining bona fide cancer neoepitopes that elicit immune-mediated cancer rejection

• Text
• PDF

Abstract

Identification of neoepitopes that are effective in cancer therapy is a major challenge in creating cancer vaccines. Here, using an entirely unbiased approach, we queried all possible neoepitopes in a mouse cancer model and asked which of those are effective in mediating tumor rejection, and independently, in eliciting a measurable CD8 response. This analysis uncovered a large trove of effective anticancer neoepitopes which have strikingly different properties from conventional epitopes and suggested an algorithm to predict them. It also revealed that our current methods of prediction discard the overwhelming majority of true anticancer neoepitopes. These results from a single mouse model were validated in another, antigenically distinct mouse cancer model, and are consistent with data reported in human studies. Structural modeling showed how the MHC I-presented neoepitopes have an altered conformation, higher stability, or increased exposure to T cell receptors as compared to the un-mutated counterparts. T cells elicited by the active neoepitopes identified here demonstrated a stem-like early dysfunctional phenotype associated with effective responses against viruses and tumors of transgenic mice. These abundant anticancer neoepitopes, which have not been tested in human studies thus far, can be exploited for the generation of personalized human cancer vaccines.

Authors

Cory A. Brennick, Mariam M. George, Marmar R. Moussa, Adam T. Hagymasi, Sahar Al Seesi, Tatiana V. Shcheglova, Ryan P. Englander, Grant L.J. Keller, Jeremy L. Balsbaugh, Brian M. Baker, Andrea Schietinger, Ion I. Mandoiu, Pramod K. Srivastava

Selective glutamine metabolism inhibition in tumor cells improves anti-tumor T lymphocyte activity in triple-negative breast cancer

• Text
• PDF

Abstract

Rapidly proliferating tumor and immune cells need metabolic programs that support energy and biomass production. The amino acid glutamine is consumed by effector T cells and glutamine-addicted triple-negative breast cancer (TNBC) cells, suggesting that a metabolic competition for glutamine may exist within the tumor microenvironment, potentially serving as a therapeutic intervention strategy. Here, we report that there is an inverse correlation between glutamine metabolic genes and markers of T cell-mediated cytotoxicity in human basal-like breast cancer (BLBC) patient datasets, with increased glutamine metabolism and decreased T cell cytotoxicity associated with poor survival. We found that tumor cell-specific loss of glutaminase (GLS), a key enzyme for glutamine metabolism, improved anti-tumor T cell activation in both a spontaneous mouse TNBC model and orthotopic grafts. The glutamine transporter inhibitor V-9302 selectively blocked glutamine uptake by TNBC cells but not CD8+ T cells, driving synthesis of GSH, a major cellular antioxidant, to improve CD8+ T cell effector function. We propose a “glutamine steal” scenario, in which cancer cells deprive tumor-infiltrating lymphocytes of needed glutamine, thus impairing anti-tumor immune responses. Therefore, tumor-selective targeting of glutamine metabolism may be a promising therapeutic strategy in TNBC.

Authors

Deanna N. Edwards, Verra M. Ngwa, Ariel L. Raybuck, Shan Wang, Yoonha Hwang, Laura C. Kim, Sung Hoon Cho, Yeeun Paik, Qingfei Wang, Siyuan Zhang, H. Charles Manning, Jeffrey C. Rathmell, Rebecca S. Cook, Mark R. Boothby, Jin Chen

Streptococcuspyogenes infects human endometrium by limiting the innate immune response

• Text
• PDF

Abstract

Group A Streptococcus (GAS), a Gram-positive human-specific pathogen yields 517,000 deaths annually worldwide, including 163,000 due to invasive infections and among them puerperal fever. Before efficient prophylactic measures were introduced, the mortality rate for mothers during childbirth was about 10%; puerperal fever still accounts for over 75,000 maternal deaths annually. Yet little is known regarding the factors and mechanisms of GAS invasion and establishment in postpartum infection. We characterized the early steps of infection in an ex vivo infection model of the human decidua, the puerperal fever portal of entry. Coordinate analysis of GAS behavior and the immune response led us to demonstrate that (i) GAS growth was stimulated by tissue products; (ii) GAS invaded tissue and killed ~50% of host cells within two hours; these processes required SpeB protease and Streptolysin O activities, respectively; (iii) GAS impaired the tissue immune response. Immune impairment occurred both at the RNA level, with only partial induction of the innate immune response, and protein level, in an SLO- and SpeB-dependent manner. Our study indicates that efficient GAS invasion of decidua and the restricted host immune response favored its propensity to develop rapid invasive infections in a gynecological-obstetrical context.

Authors

Antonin Weckel, Thomas Guilbert, Clara Lambert, Céline Plainvert, Francois Goffinet, Claire Poyart, Céline Méhats, Agnès Fouet

PD-1 blockade improves Kupffer cell bacterial clearance in acute liver injury

• Text
• PDF

Abstract

Acute liver failure (ALF) patients display systemic innate immune suppression and increased susceptibility to infections. PD-1 expression by macrophages has been associated with immune suppression during sepsis and cancer. We therefore examined the role of PD-1/PD-L1 pathway in regulating Kupffer cell inflammatory and antimicrobial responses in acetaminophen (APAP) induced acute liver injury. Using intravital imaging and flow cytometry we found impaired Kupffer cell bacterial clearance and systemic bacterial dissemination in mice with liver injury. Increased PD-1 and PD-L1 expression was detected in Kupffer cells and lymphocyte subsets, respectively, during resolution of injury. Gene expression profiling of PD-1+ Kupffer cells revealed an immune-suppressive profile and reduced pathogen responses. Compared to wild-type, PD-1 deficient or anti-PD-1 treated mice with liver injury showed improved Kupffer cell bacterial clearance, reduced tissue bacterial load and protection from sepsis. Blood sample analyses of ALF patients revealed enhanced PD-1 and PD-L1 expression of monocytes and lymphocytes, respectively, and that plasma soluble PD-L1 levels predict patient outcome and sepsis. PD-1 in vitro blockade restored monocyte functionality. Our study describes a role for PD-1/PD-L1 axis in suppressing Kupffer cell and monocyte antimicrobial responses after liver injury and suggests anti-PD-1 immunotherapy as a strategy to reduce infection susceptibility in ALF.

Authors

Evangelos Triantafyllou, Cathrin L. C. Gudd, Marie-Anne Mawhin, Hannah C. Husbyn, Francesca M. Trovato, Matthew K. Siggins, Thomas O'Connor, Hiromi Kudo, Sujit K. Mukherjee, Julia A. Wendon, Christine Bernsmeier, Robert D. Goldin, Marina Botto, Wafa Khamri, Mark J.W. McPhail, Lucia A. Possamai, Kevin J. Woollard, Charalambos G. Antoniades, Mark R. Thursz

Clinical, laboratory, and temporal predictors of neutralizing antibodies to SARS-CoV-2 among COVID-19 convalescent plasma donor candidates

• Text
• PDF

Abstract

Background: SARS-CoV-2-specific antibodies may protect from reinfection and disease, providing rationale for administration of plasma containing SARS-CoV-2 neutralizing antibodies (nAb) as a treatment for COVID-19. Clinical factors and laboratory assays to streamline plasma donor selection, and the durability of nAb responses, are incompletely understood. Methods: Potential convalescent plasma donors with virologically-documented SARS-CoV-2 infection were tested for serum IgG to SARS-CoV-2 spike protein S1 domain, nucleoprotein (NP), and for nAb. Results: Amongst 250 consecutive persons, including 27 (11%) requiring hospitalization, studied a median of 67 days since symptom onset, 97% were seropositive on one or more assays. Sixty percent of donors had nAb titers ≥1:80. Correlates of higher nAb titer included older age (adjusted odds ratio [AOR] 1.03/year of age, 95% CI 1.00-1.06), male sex (AOR 2.08, 95% CI 1.13-3.82), fever during acute illness (AOR 2.73, 95% CI 1.25-5.97), and disease severity represented by hospitalization (AOR 6.59, 95% CI 1.32-32.96). Receiver operating characteristic (ROC) analyses of anti-S1 and anti-NP antibody results yielded cutoffs that corresponded well with nAb titers, with the anti-S1 assay being slightly more predictive. NAb titers declined in 37 of 41 paired specimens collected a median of 98 days (range, 77-120) apart (P<0.001). Seven individuals (2.8%) were persistently seronegative and lacked T cell responses. Conclusions: Nab titers correlated with COVID-19 severity, age, and sex. Standard commercially available SARS-CoV-2 IgG results can serve as useful surrogates for nAb testing. Functional nAb levels were found to decline and a small proportion of persons recovered from COVID-19 lack adaptive immune responses.

Authors

Jim Boonyaratanakornkit, Chihiro Morishima, Stacy Selke, Danniel Zamora, Sarah A. McGuffin, Adrienne E. Shapiro, Victoria L. Campbell, Christopher L. McClurkan, Lichen Jing, Robin Gross, Janie Liang, Elena Postnikova, Steven Mazur, Vladimir V. Lukin, Anu Chaudhary, Marie K. Das, Susan L. Fink, Andrew Bryan, Alexander L. Greninger, Keith R. Jerome, Michael R. Holbrook, Terry B. Gernsheimer, Mark H. Wener, Anna Wald, David M. Koelle