Immunology
Abstract
Induction of podoplanin (PDPN) expression is a critical response of macrophages to LPS stimulation or bacterial infection in sepsis, but how this key process of TLR4-stimulated PDPN upregulation is regulated and the impact of PDPN expression on macrophage function remain elusive. Here, we determined how this process is regulated in vitro and in vivo. PDPN failed to be upregulated in TLR4 stimulated macrophages deficient in adhesion and degranulation-promoting adapter protein (ADAP), which could be rescued by the reconstitution of ADAP. A distinct PDPNhi peritoneal macrophage (PM) subset, which exhibited an M2-like phenotype and enhanced phagocytic activity, was generated in WT but not in ADAP-deficient septic mice. The blockade of PDPNhi PMs mimicked the effect of ADAP deficiency, which exacerbated sepsis. Mechanistically, BTK-mediated ADAP Y571 phosphorylation worked together with mTOR to converge on STAT3 activation for the transactivation of the PDPN promoter. Moreover, agonist activation of STAT3 profoundly potentiated the PDPNhi PM subset generation and alleviated sepsis severity in mice. Together, our findings reveal a mechanism whereby ADAP resets macrophage function by controlling the TLR4-induced upregulation of PDPN as a host innate immune defense during sepsis.
Authors
Pengchao Zhang, Xinning Wang, Xiaodong Yang, Hebin Liu
Abstract
Despite combination antiretroviral therapy (ART), HIV causes persistent gut barrier dysfunction, immune depletion, and dysbiosis. Further, ART interruption results in reservoir reactivation and rebound viremia. Both IL-21 and anti-α4β7 improve gut barrier functions, and we hypothesized combining them would synergize as a dual therapy to improve immunological outcomes in SIV-infected rhesus macaques (RMs). We found no significant differences in CD4+ T-cell reservoir size by intact proviral DNA assay. SIV rebounded in both dual-treated and control RMs following analytical therapy interruption (ATI), with time to rebound and initial rebound viremia comparable between groups; however, dual-treated RMs showed slightly better control of viral replication at the latest time points post-ATI. Additionally, following post-ATI, dual-treated RMs showed immunological benefits, including T-cell preservation and lower PD-1+ central memory T-cell (TCM) frequency. Notably, PD-1+ TCMs were associated with reservoir size, which predicted viral loads (VLs) post-ATI. Finally, 16S rRNA sequencing revealed better recovery from dysbiosis in treated animals, and the butyrate-producing Firmicute Roseburia predicted PD-1-expressing TCMs and VLs after ATI. PD-1+ TCMs and gut dysbiosis represent mechanisms of HIV persistence and pathogenesis, respectively. Therefore, combining IL-21 and anti-α4β7 may be an effective therapeutic strategy to improve immunological outcomes for people with HIV.
Authors
Samuel D. Johnson, Maria Pino, Arpan Acharya, Julien A. Clain, Deepanwita Bose, Kevin Nguyen, Justin Harper, Francois Villinger, Mirko Paiardini, Siddappa N. Byrareddy
Abstract
The mechanisms utilized by differentiating B cells to withstand highly damaging conditions generated during severe infections, like the massive hemolysis that accompanies malaria, are poorly understood. Here we demonstrate that ROCK1 regulates B cells differentiation in hostile environments replete with PAMPs (pathogen-associated molecular patterns) and high levels of heme by controlling two key heme-regulated molecules, BACH2 and Heme-regulated eIF2a kinase (HRI). ROCK1 phosphorylates BACH2 and protects it from heme-driven degradation. As B cells differentiate, furthermore, ROCK1 restrains their proinflammatory potential and helps them handle the heightened stress imparted by the presence of PAMPs and heme by controlling HRI, a key regulator of the integrated stress response and cytosolic proteotoxicity. ROCK1 controls the interplay of HRI with HSP90 and limits the recruitment of HRI and HSP90 to unique p62/SQSTM1 complexes that also contain critical kinases like mTORC1 and TBK1, and proteins involved in RNA metabolism, oxidative damage, and proteostasis like TDP-43. Thus, ROCK1 helps B cells cope with intense pathogen-driven destruction by coordinating the activity of key controllers of B cell differentiation and stress responses. These ROCK1-dependent mechanisms may be widely employed by cells to handle severe environmental stresses, and these findings may be relevant for immune-mediated and age-related neurodegenerative disorders.
Authors
Juan Rivera-Correa, Sanjay Gupta, Edd Ricker, Danny Flores-Castro, Daniel Jenkins, Stephen Vulcano, Swati P. Phalke, Tania Pannellini, Matthew M. Miele, Zhuoning Li, Nahuel Zamponi, Young-Bum Kim, Yurii Chinenov, Eugenia Giannopoulou, Leandro Cerchietti, Alessandra B. Pernis
Abstract
BACKGROUND. Previously, we demonstrated that changes in circulating tumor DNA (ctDNA) are promising biomarkers for early response prediction (ERP) to immune checkpoint inhibitors (ICI) in metastatic urothelial cancer (mUC). In this study, we investigated the value of whole blood immunotranscriptomics for ERP-ICI and integrated both biomarkers into a multimodal model to boost accuracy. METHODS. Blood samples of 93 patients were collected at baseline and after 2-6 weeks of ICI for ctDNA (N=88) and immunotranscriptome (N=79) analyses. ctDNA changes were dichotomized into increase or no increase, the latter including patients with undetectable ctDNA. For RNA model development, the cohort was split into a discovery (N=29), test (N=29) and validation set (N=21). Finally, RNA- and ctDNA-based predictions were integrated in a multimodal model. Clinical benefit (CB) was defined as progression-free survival beyond 6 months. RESULTS. Sensitivity (SN) and specificity (SP) of ctDNA increase for predicting non-CB (N-CB) was 59% and 92%, respectively. Immunotranscriptome analysis revealed upregulation of T-cell activation, proliferation and interferon signalling during treatment in the CB group, contrary to N-CB patients. Based on these differences a 10-gene RNA model was generated, reaching a SN and SP of 73% and 79% in the test and 67% and 67% in the validation set for predicting N-CB. Multimodal model integration led to superior performance with a SN and SP of 79% and 100% in the validation cohort. CONCLUSION. The combination of whole blood immunotranscriptome and ctDNA in a multimodal model showed promise for ERP-ICI in mUC and accurately identified patients with N-CB. TRIAL REGISTRATION. 2016-3060, 2020-6778 FUNDING. Eurostars grant E! 114908 - PRECISE, Paul Speth Foundation (Bullseye project)
Authors
Sandra van Wilpe, Davide Croci, Sara S. Fonseca Costa, Iris B.A.W. te Paske, Sofie H. Tolmeijer, Jolique van Ipenburg, Leonie I. Kroeze, Simona Pavan, Sylvain Monnier-Benoit, Guido Coccia, Noushin Hadadi, Irma M. Oving, Tineke J. Smilde, Theo van Voorthuizen, Marieke Berends, Mira D. Franken, Marjolijn J.L. Ligtenberg, Sahar Hosseinian Ehrensberger, Laura Ciarloni, Pedro Romero, Niven Mehra
Abstract
T cells targeting a KRAS mutation can induce durable tumor regression in some patients with metastatic epithelial cancer. It is unknown whether T cells targeting mutant KRAS that are capable of killing tumor cells can be identified from peripheral blood of patients with pancreatic cancer. We developed an in vitro stimulation approach and identified HLA-A*11:01–restricted KRAS G12V–reactive CD8+ T cells and HLA-DRB1*15:01–restricted KRAS G12V–reactive CD4+ T cells from peripheral blood of 2 out of 6 HLA-A*11:01–positive patients with pancreatic cancer whose tumors expressed KRAS G12V. The HLA-A*11:01–restricted KRAS G12V–reactive T cell receptor (TCR) was isolated and validated to specifically recognize the KRAS G12V8–16 neoepitope. While T cells engineered to express this TCR specifically recognized all 5 tested human HLA-A*11:01+ and KRAS G12V+ pancreatic cancer organoids, the recognition was often modest, and tumor cell killing was observed in only 2 out of 5 organoids. IFN-γ priming of the organoids enhanced the recognition and killing by the TCR-engineered T cells. The TCR-engineered T cells could significantly slow the growth of an established organoid-derived xenograft in immunodeficient mice. Our data suggest that this TCR has potential for use in TCR-gene therapy, but additional strategies that enhance tumor recognition by the TCR-engineered T cells likely will be required to increase clinical activity.
Authors
Xiongfei Xu, Shiwei Guo, Haihui Gu, Zhanshan Cha, Xiaohan Shi, Xiaoyi Yin, Huan Wang, Suizhi Gao, Bo Li, Lingyu Zhu, Wei Jing, Kailian Zheng, Zhuo Shao, Peng Cheng, Chunhong Zheng, Yi-Ping Shih, Yunguang Li, Baohua Qian, Dong Gao, Eric Tran, Gang Jin
Abstract
CD8+ T cells are critical for immune protection against severe COVID-19 during acute infection with SARS-CoV-2. However, the induction of antiviral CD8+ T cell responses varies substantially among infected people, and a better understanding of the mechanisms that underlie such immune heterogeneity is required for pandemic preparedness and risk stratification. In this study, we analyzed SARS-CoV-2-specific CD4+ and CD8+ T cell responses in relation to age, clinical status, and inflammation among patients infected primarily during the initial wave of the pandemic in France or Japan. We found that age-related contraction of the naive lymphocyte pool and systemic inflammation were associated with suboptimal SARS-CoV-2-specific CD4+ and, even more evidently, CD8+ T cell immunity in patients with acute COVID-19. No such differences were observed for humoral immune responses targeting the spike protein of SARS-CoV-2. We also found that the proinflammatory cytokine IL-18, concentrations of which were significantly elevated among patients with severe disease, suppressed the de novo induction and memory recall of antigen-specific CD8+ T cells, including those directed against SARS-CoV-2. These results potentially explain the vulnerability of older adults to infections that elicit a profound inflammatory response, exemplified by acute COVID-19.
Authors
Gaëlle Autaa, Laura Papagno, Takuto Nogimori, Andrea Boizard-Moracchini, Daniil Korenkov, Maeva Roy, Koichiro Suzuki, Yuji Masuta, Eoghann White, Sian Llewellyn-Lacey, Yasuo Yoshioka, Francesco Nicoli, David A. Price, Julie Dechanet-Merville, Takuya Yamamoto, Isabelle Pellegrin, Victor Appay
Abstract
Endometriosis is a chronic gynecological disease that affects 1 in 10 reproductive-aged women. Most studies investigate established disease; however, the initiation and early events in endometriotic lesion development remain poorly understood. Our study used neutrophils from human menstrual effluent from subjects with and without endometriosis for immunophenotyping, and a mouse model of endometriosis and a mouse endometriosis cell line to determine the role of neutrophils in the initiating events of endometriosis, including attachment and survival of minced endometrial pieces. In menstrual effluent from women with endometriosis, the ratio of aged and pro-angiogenic neutrophils increased compared to controls, indicating a potentially permissive pro-inflammatory microenvironment. In our endometriosis mouse model, knocking-down neutrophil recruitment with α-CXCR2 into the peritoneum decreased endometrial tissue adhesion—supported by decreased levels of myeloperoxidase and neutrophil elastase in both developing lesions and peritoneal fluid. Fibrinogen was identified as the preferred substrate for endometrial cell adhesion in an in vitro adhesion assay and in developing lesions in vivo. Together, aged and pro-angiogenic neutrophils and their secretions likely promote attachment and formation of endometriotic lesions by releasing neutrophil extracellular traps and upregulating fibrinogen expression as a provisional matrix to establish attachment and survival in the development of endometriosis lesions.
Authors
Taylor R. Wilson, Kurt R. Peterson, Stephanie A. Morris, Damaris Kuhnell, Susan Kasper, Katherine A. Burns
Abstract
Hereditary angioedema is an autosomal dominant disorder caused by defects in C1-esterase inhibitor (C1-INH), resulting in poorly controlled activation of the kallikrein-kinin system and bradykinin overproduction. C1-INH is a heavily glycosylated protein in the serine protease inhibitor (SERPIN) family, yet the role of these glycosylation sites remains unclear. To elucidate the functional impact of N-glycosylation in the SERPIN domain of C1-INH, we engineered four sets consisting of 26 variants at or near the N-linked sequon (NXS/T). Among these, six are reported in HAE patients and five are known C1-INH variants without accessible clinical histories. We systematically evaluated their expression, structure and functional activity with C1¯s, FXIIa and kallikrein. Our findings showed that of the eleven reported variants, seven are deleterious. Deleting N at the three naturally occurring N-linked sequons (N238, N253 and N352) results in pathologic consequences. Altering these sites by substituting N to A disrupts N-linked sugar attachment but preserves protein expression or function. Further, an additional N-linked sugar generated at N272 impairs C1-INH function. These findings highlight the importance of N-linked sequons in modulating the expression and function of C1-INH. Insights gained from identifying the pathological consequences of N-glycan variants should assist in defining more tailored therapy.
Authors
Zhen Ren, John Bao, Shuangxia Zhao, Nicola Pozzi, H. Wedner, John P. Atkinson
Abstract
Thrombin promotes the proliferation and function of CD8+ T cells. To test if thrombin prevents exhaustion and sustains antiviral T cell activity during chronic viral infection, we depleted the thrombin-precursor prothrombin to 10% of normal levels in mice prior to infection with the clone 13 strain of lymphocytic choriomeningitis virus. Unexpectedly, prothrombin insufficiency resulted in 100% mortality after infection that was prevented by depletion of CD8+ T cells, suggesting that reduced availability of prothrombin enhances virus-induced immunopathology. Yet, the number, function, and apparent exhaustion of virus-specific T cells were measurably unaffected by prothrombin depletion. Histological analysis of the lung, heart, liver, kidney, spleen, intestine, and brain did not reveal any evidence of hemorrhage or increased tissue damage in low prothrombin mice that could explain mortality. Viral loads were also similar in infected mice regardless of prothrombin levels. Instead, infection of prothrombin-depleted mice resulted in a severe, T cell-dependent anemia associated with increased hemolysis. Thus, thrombin plays an unexpected protective role in preventing hemolytic anemia during virus infection, with potential implications for patients who are using direct thrombin inhibitors as an anticoagulant therapy.
Authors
Rachel Cantrell, H. Alex Feldman, Leah Rosenfeldt, Ayad Ali, Benjamin Gourley, Cassandra Sprague, Daniel Leino, Jeff Crosby, Alexey Revenko, Brett Monia, Stephen N. Waggoner, Joseph S. Palumbo
Highly multiplexed imaging reveals prognostic immune and stromal spatial biomarkers in breast cancer
Abstract
Spatial profiling of tissues promises to elucidate tumor-microenvironment interactions and generate prognostic and predictive biomarkers. We analyzed single-cell, spatial data from three multiplex imaging technologies: cyclic immunofluorescence (CycIF) data we generated from 102 breast cancer patients with clinical follow-up, and publicly available imaging mass cytometry and multiplex ion-beam imaging datasets. Similar single-cell phenotyping results across imaging platforms enabled combined analysis of epithelial phenotypes to delineate prognostic subtypes among estrogen-receptor positive (ER+) patients. We utilized discovery and validation cohorts to identify biomarkers with prognostic value. Increased lymphocyte infiltration was independently associated with longer survival in triple-negative (TN) and high-proliferation ER+ breast tumors. An assessment of ten spatial analysis methods revealed robust spatial biomarkers. In ER+ disease, quiescent stromal cells close to tumor were abundant in good prognosis tumors, while tumor cell neighborhoods containing mixed fibroblast phenotypes were enriched in poor prognosis tumors. In TN disease, macrophage/tumor and B/T lymphocyte neighbors were enriched and lymphocytes were dispersed in good prognosis tumors, while tumor cell neighborhoods containing vimentin-positive fibroblasts were enriched in poor prognosis tumors. In conclusion, we generated comparable single-cell spatial proteomic data from several clinical cohorts to enable prognostic spatial biomarker identification and validation.
Authors
Jennifer R. Eng, Elmar Bucher, Zhi Hu, Cameron R. Walker, Tyler Risom, Michael Angelo, Paula Gonzalez-Ericsson, Melinda E. Sanders, A. Bapsi Chakravarthy, Jennifer A. Pietenpol, Summer L. Gibbs, Rosalie C. Sears, Koei Chin
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