Immune and inflammatory responses to SARS-CoV-2 contribute to disease severity of COVID-19. However, the utility of specific immune-based biomarkers to predict clinical outcome remains elusive. Here, we analyzed levels of 66 soluble biomarkers in 175 Italian patients with COVID-19 ranging from mild/moderate to critical severity, and assessed type-I IFN-, type-II IFN-, and NF-κB-dependent whole blood transcriptional signatures. A broad inflammatory signature was observed, implicating activation of various immune and non-hematopoietic cell subsets. Discordance between IFN-α2a protein and IFNA2 transcript levels in blood suggests that type-I IFNs during COVID-19 may be primarily produced by tissue-resident cells. Multivariable analysis of patients’ first samples revealed 12 biomarkers (CCL2, IL-15, sST2, NGAL, sTNFRSF1A, ferritin, IL-6, S100A9, MMP-9, IL-2, sVEGFR1, IL-10) that when increased were independently associated with mortality. Multivariate analyses of longitudinal biomarker trajectories identified 8 of the aforementioned biomarkers (IL-15, IL-2, NGAL, CCL2, MMP-9, sTNFRSF1A, sST2, IL-10) and two additional biomarkers (lactoferrin, CXCL9) that were significantly associated with mortality when increased, while IL-1α was associated with mortality when decreased. Among these, sST2, sTNFRSF1A, IL-10, and IL-15 were consistently higher throughout the hospitalization in patients who died versus those who recovered, suggesting that these biomarkers may provide an early warning of eventual disease outcome.
Michael S. Abers, Ottavia M. Delmonte, Emily E. Ricotta, Jonathan Fintzi, Danielle Fink, Adriana A. de Jesus, Kol A. Zarember, Sara Alehashemi, Vasileios Oikonomou, Jigar V. Desai, Scott W. Canna, Bita Shakoory, Kerry Dobbs, Luisa Imberti, Alessandra Sottini, Eugenia Quiros-Roldan, Francesco Castelli, Camillo Rossi, Duilio Brugnoni, Andrea Biondi, Laura R. Bettini, Mariella D’Angio’, Paolo Bonfanti, Riccardo Castagnoli, Daniela Montagna, Amelia Licari, Gian Luigi Marseglia, Emily Gliniewicz, Elana R. Shaw, Dana Kahle, Andre T. Rastegar, Michael A Stack, Katherine Myint-Hpu, Susan L. Levinson, Mark J. DiNubile, Daniel W. Chertow, Peter Burbelo, Jeffrey I. Cohen, Katherine R. Calvo, John S. Tsang, Helen C. Su, John I. Gallin, Douglas B. Kuhns, Raphaela Goldbach-Mansky, Michail S Lionakis, Luigi D Notarangelo
Glioblastoma multiforme (GBM) is a fatal human cancer in part because GBM stem cells are resistant to therapy and recurrence is inevitable. Previously, we demonstrated Zika virus (ZIKV) targets GBM stem cells and prevents death of mice with gliomas. Here, we evaluated the immunological basis of ZIKV-mediated protection against GBM. Introduction of ZIKV into the brain tumor increases recruitment of CD8+ T and myeloid cells to the tumor microenvironment. CD8+ T cells are required for ZIKV-dependent tumor clearance, as survival benefits are lost with CD8+ T cell depletion. Moreover, while anti-PD1 antibody therapy alone moderately improves tumor survival, when co-administered with ZIKV, survival increases. ZIKV-mediated tumor clearance also results in durable protection against syngeneic tumor re-challenge, which also depends on CD8+ T cells. To address safety concerns, we generated an immune-sensitized ZIKV strain, which is effective alone or in combination with immunotherapy. Thus, oncolytic ZIKV treatment can be leveraged by immunotherapies, which may prompt combination treatment paradigms for adult GBM patients.
Sharmila Nair, Luciano Mazzoccoli, Arijita Jash, Jennifer Govero, Sachendra S. Bais, Tong Hu, Camila R. Fontes-Garfias, Chao Shan, Hideho Okada, Sujan Shresta, Jeremy N. Rich, Pei-Yong Shi, Michael S. Diamond, Milan G. Chheda
The nonimmune roles of Tregs have been described in various tissues, including the BM. In this study, we comprehensively phenotyped marrow Tregs, elucidating their key features and tissue-specific functions. We show that marrow Tregs are migratory and home back to the marrow. For trafficking, marrow Tregs use S1P gradients, and disruption of this axis allows for specific targeting of the marrow Treg pool. Following Treg depletion, the function and phenotype of both mesenchymal stromal cells (MSCs) and hematopoietic stem cells (HSCs) was impaired. Transplantation also revealed that a Treg-depleted niche has a reduced capacity to support hematopoiesis. Finally, we found that marrow Tregs are high producers of IL-10 and that Treg-secreted IL-10 has direct effects on MSC function. This is the first report to our knowledge revealing that Treg-secreted IL-10 is necessary for stromal cell maintenance, and our work outlines an alternative mechanism by which this cytokine regulates hematopoiesis.
Virginia Camacho, Victoria R. Matkins, Sweta B. Patel, Jeremie M. Lever, Zhengqin Yang, Li Ying, Ashley E. Landuyt, Emma C. Dean, James F. George, Henry Yang, Paul Brent Ferrell, Craig L. Maynard, Casey T. Weaver, Heth R. Turnquist, Robert S. Welner
Congenital Zika syndrome (CZS) is associated with microcephaly and various neurological, musculoskeletal, and ocular abnormalities, but the long-term pathogenesis and postnatal progression of ocular defects in infants are not well characterized. Rhesus macaques are superior to rodents as models of CZS because they are natural hosts of the virus and share similar immune and ocular characteristics, including blood-retinal barrier characteristics and the unique presence of a macula. Using a previously-described model of CZS by infecting pregnant rhesus macaques with Zika virus (ZIKV) during the late first trimester, we characterized postnatal ocular development and evolution of ocular defects in 2 infant macaques over 2 years. We found that one of these animals exhibited colobomatous chorioretinal atrophic lesions with macular and vascular dragging, as well as retinal thinning caused by loss of retinal ganglion neuron and photoreceptor layers. Despite these congenital ocular malformations, axial elongation and retinal development in these infants progressed at normal rates compared to healthy animals. The ZIKV-exposed infants displayed a rapid loss of ZIKV-specific antibodies, suggesting the absence of viral replication after birth, and did not show any behavioral or neurological defects postnatally. Our findings suggest that ZIKV infection during early pregnancy can impact fetal retinal development and cause congenital ocular anomalies, but does not appear to affect postnatal ocular growth.
Glenn Yiu, Sara M. Thomasy, M. Isabel Casanova, Alexander M. Rusakevich, Rebekah I. Keesler, Jennifer Watanabe, Jodie Usachenko, Anil Singapuri, Erin E. Ball, Eliza Bliss-Moreau, Wendi Guo, Helen Webster, Tulika Singh, Sallie R. Permar, Amir Ardeshir, Lark L. Coffey, Koen K.A. Van Rompay
Chimeric antigen receptor (CAR) T cell therapy for solid tumors has shown limited efficacy in early-phase clinical studies. The majority of CARs encode CD28 and/or 41BB costimulatory endodomains, and we explored whether MyD88 and CD40 (MC) costimulatory endodomains in CARs could improve their antitumor activity. We generated CD28-, 41BB-, and MC-CAR T cells and demonstrated that MC-CAR T cells have greater proliferative capacity and antitumor activity in repeat stimulation assays and in tumor models in vivo. Transcriptomic analysis revealed that MC-CAR T cells expressed higher levels of MYB and FOXM1, key cell cycle regulators, and were activated at baseline. After stimulation, MC-CAR T cells remained in a less differentiated state than CD28- and 41BB-CAR T cells as judged by low levels of transcription factor TBET and B lymphocyte induced maturation protein 1 expression and lower cytolytic activity in comparison with CD28- and 41BB-CAR T cells. Thus, including MyD88 and CD40 signaling domains in CARs may improve current CAR T cell therapy approaches for solid tumors.
Brooke Prinzing, Patrick Schreiner, Matthew Bell, Yiping Fan, Giedre Krenciute, Stephen Gottschalk
Alveolar macrophages (AM) are differentially regulated by human surfactant protein-A (SP-A)1 or SP-A2. However, AM are very heterogeneous and differences are difficult to characterize in intact cells. Using the Toponome Imaging System (TIS), an imaging technique that uses sequential immunostaining to identity patterns of biomarker expression or combinatorial molecular phenotypes (CMP), we studied individual single cells and identified subgroups of AM (n=168) from SP-A knockout (KO) mice and mice expressing either SP-A1 or SP-A2. The effects, as shown by CMPs, of SP-A1 and SP-A2 on AM were significant and differed. SP-A1 AM were the most diverse and shared the fewest CMPs with KO and SP-A2. Clustering analysis of each group showed three clusters where the CMP-based phenotype was distinct in each cluster. Moreover, a clustering analysis of all 168 AM revealed ten clusters, many dominated by one group. Some CMP, overlap among groups was observed with SP-A2 AM sharing the most CMPs and SP-A1 AM the fewest. The CMP-based patterns identified here provide a basis for not only understanding AM diversity, but, most importantly, the molecular basis for the diversity of functional differences in mouse models where the impact of genetics of innate immune molecules on AM has been studied.
David S. Phelps, Vernon M. Chinchilli, Judith Weisz, Lili Yang, Debra Shearer, Xuesheng Zhang, Joanna Floros
A possible etiological link between the onset of endemic pemphigus in Tunisia and bites of Phlebotomus (P). papatasi, the vector of zoonotic cutaneous leishmaniasis has been previously suggested. We hypothesized that the immunodominant P. papatasi salivary protein PpSP32 binds to desmogleins (Dsg1 and Dsg3), triggering loss of tolerance to these pemphigus target autoantigens. We here show by Far-Western blot that the recombinant PpSP32 protein (rPpSP32) binds to epidermal proteins with a molecular weight of about 170kDa. Co-immunoprecipitation revealed the interaction of rPpSP32 with either Dsg1 or Dsg3. A specific interaction between PpSP32 and Dsg (1 and 3) was further demonstrated by ELISA assays. Finally, mice immunized with rPpSP32 twice a week exhibited significantly increased levels of anti-Dsg1 and anti-Dsg3 antibodies from day 75 to day 120. Such antibodies were specific for Dsg1 and Dsg3 and were not the result of crossreactivity to PpSP32. Herein, we demonstrated for the first time a specific binding between the PpSP32 and the dsg1 and 3, which might underlie the triggering of anti-Dsg antibodies in patients exposed to sand fly bites. We also confirmed the development of specific anti-Dsg1 and -Dsg3 antibodies in vivo after PpSP32 immunization in mice. Collectively, our results provide evidence that environmental factors, such the exposure to P. papatasi bites, can trigger the development of autoimmune antibodies.
Soumaya Marzouki, Ines Zaraa, Maha Abdeladhim, Chaouki Ben Abdessalem, Fabiano Oliveira, Shaden Kamhawi, Mourad Mokni, Hechmi Louzir, Jesus Valenzuela, Mélika Ben Ahmed
Plasma antimalarial antibody can mediate anti-parasite immunity but has not previously been characterized at the molecular level. Here, we develop an innovative strategy to characterize humoral responses by integrating profiles of plasma immunoglobulins (IG) or antibodies with those expressed on B cells as part of BcR. We applied this strategy to define plasma IG and determine variable V gene usage after vaccination with the Plasmodium falciparum zygote antigen Pfs25. First, using proteomic tools coupled with bulk immunosequencing data, we determined human F(ab′)2 peptide sequences from plasma IG of adults who received four doses of Pfs25-EPA/Alhydrogel. Specifically, Pfs25 antigen-specific F(ab′)2 peptides (Pfs25-IG) were aligned to cDNA sequences of IGH complementarity determining region 3 (CDR3) from a dataset generated by total peripheral B cell immunosequencing of the entire vaccinated population. IGHV4 was the most commonly identified IGHV subgroup of Pfs25-IG, a pattern that was corroborated by VH/VL sequencing of Pfs25-specific single B cells from five vaccinees and by matching plasma Pfs25-IG peptides and V-(D)-J sequences of Pfs25-specific single B cells from the same donor. Among 13 recombinant human mAbs generated from IG sequences of Pfs25-specific single B cells, a single IGHV4 mAb displayed strong neutralizing activity, reducing the number of P. falciparum oocysts in infected mosquitoes by more than 80% at 100 μg/mL. Our approach characterizes the human plasma antibody repertoire in response to the Pfs25-EPA/Alhydrogel vaccine and will be useful to study circulating antibodies in response to other vaccines as well as those induced during infections or autoimmune disorders.
Camila H. Coelho, Steven T. Nadakal, Patricia A. Gonzales Hurtado, Robert Morrison, Jacob D. Galson, Jillian Neal, Yimin Wu, C. Richter King, Virginia Price, Kazutoyo Miura, Sharon Wong-Madden, Justin Y.A. Dortichamou, David L. Narum, Nicholas J. MacDonald, Maryonne Snow-Smith, Marissa Vignali, Justin J. Taylor, Marie-Paule Lefranc, Johannes Trück, Carole A. Long, Issaka Sagara, Michal Fried, Patrick E. Duffy
Engineering T cells to express chimeric antigen receptors (CARs) specific for antigens on hematological cancers has yielded remarkable clinical responses, but with solid tumors, benefit has been more limited. This may reflect lack of suitable target antigens, immune evasion mechanisms in malignant cells, and/or lack of T cell infiltration into tumors. An alternative approach, to circumvent these problems, is targeting the tumor vasculature rather than the malignant cells directly. CLEC14A is a glycoprotein selectively overexpressed on the vasculature of many solid human cancers and is, therefore, of considerable interest as a target antigen. Here, we generated CARs from 2 CLEC14A-specific antibodies and expressed them in T cells. In vitro studies demonstrated that, when exposed to their target antigen, these engineered T cells proliferate, release IFN-γ, and mediate cytotoxicity. Infusing CAR engineered T cells into healthy mice showed no signs of toxicity, yet these T cells targeted tumor tissue and significantly inhibited tumor growth in 3 mouse models of cancer (Rip-Tag2, mPDAC, and Lewis lung carcinoma). Reduced tumor burden also correlated with significant loss of CLEC14A expression and reduced vascular density within malignant tissues. These data suggest the tumor vasculature can be safely and effectively targeted with CLEC14A-specific CAR T cells, offering a potent and widely applicable therapy for cancer.
Xiaodong Zhuang, Federica Maione, Joseph Robinson, Michael Bentley, Baksho Kaul, Katharine Whitworth, Neeraj Jumbu, Elizabeth Jinks, Jonas Bystrom, Pietro Gabriele, Elisabetta Garibaldi, Elena Delmastro, Zsuzsanna Nagy, David Gilham, Enrico Giraudo, Roy Bicknell, Steven P. Lee
The integration of HIV DNA into the host genome contributes to lifelong infection in most individuals. Few studies have examined integration in lymphoid tissue, where HIV predominantly persists before and after antiretroviral treatment (ART). Of particular interest is whether integration site distributions differ between infection stages with paired blood and tissue comparisons. Here, we profiled HIV integration site distributions in sorted memory, tissue resident, and/or follicular helper CD4+ T-cell subsets from paired blood and lymphoid tissue samples from acute, chronic, and ART-treated individuals (n=3 each). We observed minor differences in the frequency of non-intronic and non-distal intergenic sites varying with tissue and residency phenotypes during ART. Genomic and epigenetic annotations were generally similar. Clonal expansion of cells marked by identical integration sites was detected, with increased detection in chronic and ART-treated individuals. However, overlap between or within CD4+ T-cell subsets or tissue compartments was only observed in 8 unique sites out of 3,540 sites studied. Together, these findings suggest that shared integration sites between blood and tissue may, depending on the tissue site, be the exception rather than the rule, and indicate that additional studies are necessary to fully understand the heterogeneity of tissue sequestered HIV reservoirs.
Vincent H. Wu, Christopher L Nobles, Leticia Kuri-Cervantes, Kevin McCormick, John K. Everett, Son Nguyen, Perla M. del Río Estrada, Mauricio González-Navarro, Santiago Ávila-Ríos, Gustavo Reyes-Terán, Frederic D. Bushman, Michael R. Betts
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