Insight into processes that determine CD8+ T cell memory formation has been obtained from infection models. These models are biased toward an inflammatory milieu and often employ high avidity CD8+ T cells in adoptive transfer procedures. It is unclear whether these conditions mimic the differentiation processes of an endogenous repertoire that proceed upon non-inflammatory conditions prevailing in premalignant tumor lesions. We examined the role of cytolytic capacity on CD8+ T cell fate decisions when primed by tumor cells or by minor histocompatibility antigen-mismatched leukocytes. CD8+ memory commitment was analyzed in Ebag9-deficient mice that exhibit an enhanced tumor cell lysis. This property endowed Ebag9-/- mice with extended control of Tcl-1 oncogene-induced chronic lymphocytic leukemia progression. In Ebag9-/- mice, an expanded memory population was obtained for anti-HY and anti-SV40 T antigen-specific T cells, despite unchanged effector frequencies in the primary response. By comparing the single-cell transcriptomes of CD8+ T cells responding to tumor cell vaccination, we found differential distribution of subpopulations between Ebag9+/+ and Ebag9-/- T cells. In Ebag9-/- cells, these larger clusters contained genes encoding transcription factors regulating memory cell differentiation, along with anti-apoptotic gene functions. Our findings link EBAG9-controlled cytolytic activity and the commitment to the CD8+ memory lineage.
Armin Rehm, Anthea Wirges, Dana Hoser, Cornelius Fischer, Stefanie Herda, Kerstin Gerlach, Sascha Sauer, Gerald Willimsky, Uta E. Höpken
Secondary infections are frequent complications of viral respiratory infections but the potential consequence of SARS-CoV-2 co-infection with common pulmonary pathogens is poorly understood. We report that co-infection of human ACE2 transgenic mice with sublethal doses of SARS-CoV-2 and Streptococcus pneumoniae results in synergistic lung inflammation and lethality. Mortality was observed regardless of whether SARS-CoV-2 challenge occurred before or after establishment of sublethal pneumococcal infection. Increased bacterial levels following co-infection were associated with alveolar macrophage depletion and treatment with murine GM-CSF reduced lung bacteria numbers and pathology, and partially protected from death. However, therapeutic targeting of interferons, an approach that is effective against influenza co-infections, failed to increase survival. Combined vaccination against both SARS-CoV-2 and pneumococci resulted in 100% protection against subsequent co-infection. The results indicate that when seasonal respiratory infections return to pre-pandemic levels, they could lead to an increased incidence of lethal COVID-19 superinfections, especially among the unvaccinated population.
Tarani Kanta Barman, Amit K. Singh, Jesse L. Bonin, Tanvir N. Nafiz, Sharon L. Salmon, Dennis W. Metzger
BACKGROUND. Fibrolamellar carcinoma (FLC) is an aggressive liver cancer primarily afflicting adolescents and young adults. Patients with FLC harbor a heterozygous deletion on chromosome 19 that leads to the oncogenic gene fusion, DNAJB1-PRKACA. There are currently no effective therapeutics for FLC. To work toward that end, it is critical to gain deeper mechanistic insight into FLC pathogenesis. METHODS. We assembled a large sample set of FLC and non-malignant liver tissue (n=52) and performed integrative multi-omic analysis. Specifically, we carried out small RNA-sequencing to define altered microRNA expression patterns in tumor samples and then coupled this analysis with RNA sequencing (RNA-seq) and chromatin run-on sequencing (ChRO-seq) data to identify candidate master microRNA regulators of gene expression in FLC. We also evaluated the relationship between DNAJB1-PRKACA and microRNAs of interest in several human and mouse cell models. Finally, we performed loss-of-function experiments for a specific microRNA in cells established from a patient-derived xenograft (PDX) model. RESULTS. We identified miR-10b-5p and miR-455-3p as the top candidate pro-proliferative and tumor suppressive microRNAs, respectively. In multiple human cell models, but not in mouse cell models, over-expression of DNAJB1-PRKACA leads to significant up-regulation of miR-10b-5p. In cells established from a PDX model, inhibition of miR-10b increases the expression of several novel target genes, including the anti-proliferative factors TRIM35 and SUN2, concomitant with a significant reduction in metabolic activity, anchorage-independent growth, and proliferation. CONCLUSION. This functional genomics study highlights a novel proliferative axis in FLC and provides a rich resource for further investigation of the molecular landscape of FLC. The results reveal that miR-10b-5p shapes gene expression and promotes cell proliferation in FLC. Future studies are necessary to identify how the loss of miR-455-3p contributes to FLC progression and how miR-10b-5p may coordinate with miR-455-3p to control tumor phenotypes.
Adam B. Francisco, Matt Kanke, Andrew P. Massa, Timothy A. Dinh, Ramja Sritharan, Khashayar Vakili, Nabeel Bardeesy, Praveen Sethupathy
Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a chronic, debilitating multisystem illness of unknown etiology for which there is no cure and no diagnostic tests available. Despite increasing evidence implicating EBV and human herpesvirus-6A (HHV-6A) as potential causative infectious agents in a subset of ME/CFS patients, there are few mechanistic studies to address a causal relationship. In this study we examined a large ME/CFS cohort (n=351) and 77 controls and demonstrate a significant increase in activin A and IL-21serum levels, which correlated with seropositivity for antibodies to the EBV and HHV-6 protein deoxyuridine-triphosphate nucleotidohydrolase (dUTPase), but not CXCL13. These cytokines are critical for T follicular helper (TFH) cell differentiation, generation of high-affinity antibodies and long-lived plasma cells. Notably, ME/CFS serum was sufficient to drive TFH cell differentiation via an activin A-dependent mechanism. The lack of simultaneous CXCL13 increase with IL-21 indicates impaired TFH-function in ME/CFS. In vitro studies revealed that virus-dUTPases strongly induced activin A secretion while in vivo, EBV-dUTPase induced the formation of splenic marginal zone B and invariant NKTFH cells. Altogether, our data indicate abnormal germinal center (GC) activity in ME/CFS subjects and highlight a mechanism by which EBV and HHV6-dUTPases may alter GC and extrafollicular Ab responses.
Brandon S. Cox, Khaled Alharshawi, Irene Mena-Palomo, William P. Lafuse, Maria E. Ariza
Rhodopsin (RHO)-associated retinitis pigmentosa (RP) is a progressive retinal disease that currently has no cure. RHO protein misfolding leads to disturbed proteostasis and the death of rod photoreceptors, resulting in decreased vision. We previously identified non-retinoid chaperones of RHO, including YC-001 and F5257-0462, by small-molecule high-throughput screening. Here, we profile the chaperone activities of these molecules towards the cell-surface level of 27 RP-causing human RHO mutants in NIH3T3 cells. Further, using retinal explant culture, we show that YC-001 improves retinal proteostasis by supporting RHO homeostasis in RhoP23H/+ mouse retinae, which results in thicker outer nuclear layers (ONL) indicating delayed photoreceptor degeneration. Interestingly, YC-001 ameliorated retinal immune responses and reduced the number of microglia/macrophages in the RhoP23H/+ retinal explants. Similarly, F5257-0462 also protects photoreceptors in RhoP23H/+ retinal explants. In vivo, intravitreal injection of YC-001 or F5257-0462 microparticles in PBS shows that F5257-0462 has a higher efficacy in preserving photoreceptor function and delaying photoreceptor death in RhoP23H/+ mice. Collectively, we provide proof of principle that non-retinoid chaperones are promising drug candidates in treating RHO-associated RP.
Abhishek Vats, Yibo Xi, Bing Feng, Owen D. Clinger, Anthony J. St. Leger, Xujie Liu, Archisha Ghosh, Chase D. Dermond, Kira L. Lathrop, Gregory P. Tochtrop, Serge Picaud, Yuanyuan Chen
Hvcn1 is a voltage-gated proton channel, which reduces cytosol acidification and facilitates the production of reactive oxygen species (ROS). The increased expression of this channel in some cancers, has led to proposing Hvcn1 antagonists as potential therapeutics.While its role in most leukocytes has been studied in-depth, the function of Hvcn1 in T-cells remains poorly defined. We show that HVCN1 plays a non-redundant role in protecting naïve T-cells from intracellular acidification during priming. Despite sharing overall functional impairment in vivo and in vitro, Hvcn1-deficient CD4+ and CD8+ T-cells display profound differences during the transition from naïve to primed T-cells, including in the preservation of TCR signaling, cellular division and death. These selective features result, at least in part, from a substantially different metabolic response to intracellular acidification associated with priming. While Hvcn1-deficient naïve CD4+ T-cells reprogram to rescue the glycolytic pathway, naïve CD8+ T-cells, which express high levels of this channel in the mitochondria, respond by metabolically compensating mitochondrial dysfunction, at least in part via AMPK activation.These observations imply heterogeneity between adaptation of naïve CD4+ and CD8+ T-cells to intracellular acidification during activation.
David Coe, Thanushiyan Poobalasingam, Hongmei Fu, Fabrizia Bonacina, Guosu Wang, Valle Morales, Annalisa Moregola, Nico Mitro, Kenneth C.P. Cheung, Eleanor J. Ward, Suchita Nadkarni, Dunja Aksentijevic, Katiuscia Bianchi, Giuseppe Danilo Norata, Melania Capasso, Federica M. Marelli-Berg
BACKGROUND. Limited information is available on the impact of immunosuppressants on COVID-19 vaccination in patients with immune-mediated inflammatory diseases (IMID). METHODS. This observational cohort study examined the immunogenicity of SARS-CoV-2 mRNA vaccines in adult patients with inflammatory bowel disease, rheumatoid arthritis, ankylosing spondylitis, or psoriatic disease, with or without maintenance immunosuppressive therapies. Antibody and T cell responses to SARS-COV-2, including neutralization against SARS-CoV-2 variants were determined before and after 1 and 2 vaccine doses. RESULTS. We prospectively followed 150 subjects, 26 healthy controls, 9 IMID patients on no treatment, 44 on anti-TNF, 16 on anti-TNF with methotrexate/azathioprine (MTX/AZA), 10 on anti-IL-23, 28 on anti-IL-12/23, 9 on anti-IL-17, and 8 on MTX/AZA. Antibody and T cell responses to SARS-CoV-2 were detected in all participants, increasing from dose 1 to dose 2 and declining 3 months later, with greater attrition in IMID patients compared to healthy controls. Antibody levels and neutralization efficacy against variants of concern were substantially lower in anti-TNF treated patients than in healthy controls and were undetectable against Omicron by 3 months after dose 2. CONCLUSIONS. Our findings support the need for a third dose of mRNA vaccine and for continued monitoring of immunity in these patient groups. FUNDING. Funded by a donation from Juan and Stefania Speck and by Canadian Institutes of Health (CIHR) /COVID-Immunity Task Force (CITF) grants VR-1 172711 and VS1-175545 (T.H.W. and A.C.G); CIHR FDN-143250 (T.H.W.), GA2- 177716 (V.C., A.C.G., T.H.W.), GA1-177703 (A.C.G.) and the CIHR rapid response network to SARS-CoV-2 variants, CoVaRR-Net (to A.C.G.).
Roya M. Dayam, Jaclyn C. Law, Rogier L. Goetgebuer, Gary Y.C. Chao, Kento T. Abe, Mitchell Sutton, Naomi Finkelstein, Joanne M. Stempak, Daniel Pereira, David Croitoru, Lily Acheampong, Saima Rizwan, Klaudia Rymaszewski, Raquel Milgrom, Darshini Ganatra, Nathalia V. Batista, Melanie Girard, Irene Lau, Ryan Law, Michelle W. Cheung, Bhavisha Rathod, Julia Kitaygorodsky, Reuben Samson, Queenie Hu, W. Rod Hardy, Nigil Haroon, Robert D. Inman, Vincent Piguet, Vinod Chandran, Mark S. Silverberg, Anne-Claude Gingras, Tania H. Watts
COVID-19 infection causes collapse of glomerular capillaries and loss of podocytes, terminating in a severe kidney disease called COVID-19 associated nephropathy (COVAN). The underlying mechanism of COVAN is unknown. We hypothesized that cytokines induced by COVID-19 trigger expression of pathogenic APOL1 via JAK-STAT signaling, resulting in podocyte loss and COVAN phenotype. Here, based on nine biopsy-proven COVAN cases, we demonstrated for the first time that APOL1 protein is abundantly expressed in podocytes and glomerular endothelial cells (GECs) of COVAN kidneys but not in controls. Moreover, a majority (77.8%) of COVAN patients carried two APOL1 risk alleles. We showed that recombinant cytokines induced by SARS-CoV-2 act synergistically to drive APOL1 expression through the JAK-STAT pathway in primary human podocytes, GECs, and kidney micro-organoids derived from a carrier of two APOL1 risk alleles but was blocked by JAK1/2-inhibitor, baricitinib. We demonstrated for the first time that cytokine-induced JAK-STAT-APOL1 signaling reduced the viability of kidney organoid podocytes but was rescued by baricitinib. Together, our results support the conclusion that COVID-19-induced cytokines are sufficient to drive COVAN-associated podocytopathy via JAK-STAT-APOL1 signaling and that JAK-inhibitor could block this pathogenic process. These findings suggest that JAK-inhibitors may have therapeutic benefits for managing cytokine-induced APOL1-mediated podocytopathy.
Sarah E. Nystrom, Guojie Li, Somenath Datta, Karen Soldano, Daniel Silas, Astrid Weins, Gentzon Hall, David B. Thomas, Opeyemi A. Olabisi
K-ras mutant lung adenocarcinoma (KM-LUAD) is associated with abysmal prognosis and tightly linked to tumor-promoting inflammation. A human monoclonal antibody targeting proinflammatory cytokine IL-1β, Canakinumab, was found to significantly decrease the risk of lung cancer in the Canakinumab Anti-inflammatory Thrombosis Outcomes Study. Interestingly, we found high levels of IL-1β in the lungs of mice with K-rasG12D mutant tumors (CC-LR). Here we blocked IL-1β using a monoclonal anti-IL-1β antibody in cohorts of 6 or 14-week-old CC-LR mice to explore its preventive and therapeutic effect, respectively. IL-1β blockade significantly reduced lung tumor burden, which was associated with reprogramming of the lung microenvironment toward an anti-tumor phenotype characterized by increased infiltration of cytotoxic CD8+ T cells (with high IFN-γ and granzyme B but low PD-1 expression) while suppressing neutrophil and polymorphonuclear myeloid-derived suppressor cell responses. In querying the TCGA dataset, we interestingly found positive correlations between IL1B expression and infiltration of immunosuppressive polymorphonuclear cells and expression of their chemoattractant, CXCL1, and PDCD1 expressions in patients with KM-LUAD. Our data provide evidence that IL-1β blockade may serve as a preventive strategy among high-risk individuals and an alternative therapeutic approach in combination with currently available treatments for KM-LUAD.
Bo Yuan, Michael J. Clowers, Walter V. Velasco, Stephen Peng, Qian Peng, Yewen Shi, Marco Ramos-Castaneda, Melody Zarghooni, Shuanying Yang, Rachel L. Babcock, Seon Hee Chang, John V. Heymach, Jianjun Zhang, Edwin J. Ostrin, Stephanie S. Watowich, Humam Kadara, Seyed Javad Moghaddam
Inflammation of the esophageal epithelium is a hallmark of eosinophilic esophagitis (EoE), an emerging chronic allergic disease. Herein, we probed human esophageal epithelial cells at single-cell resolution during homeostasis and EoE. During allergic inflammation, the epithelial differentiation program was blocked, leading to loss of KRT6high differentiated populations and expansion of TOP2high proliferating and DSPhigh, SERPINB3high transitioning populations; however, there was stability of the stem cell–enriched PDPNhigh basal epithelial compartment. This differentiation program blockade was associated with dysregulation of transcription factors, including nuclear receptor signalers, in the most differentiated epithelial cells and altered NOTCH-related cell-to-cell communication. Each epithelial population expressed genes with allergic disease risk variants, supporting their functional interplay. The esophageal epithelium differed notably between EoE in histologic remission and controls, indicating that remission is a transitory state poised to relapse. Collectively, our data uncover the dynamic nature of the inflamed human esophageal epithelium and provide a framework to better understand esophageal health and disease.
Mark Rochman, Ting Wen, Michael Kotliar, Phillip J. Dexheimer, Netali Ben-Baruch Morgenstern, Julie M. Caldwell, Hee-Woong Lim, Marc E. Rothenberg
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