Severe COVID-19 disease is associated with dysregulation of the myeloid compartment during acute infection. Survivors frequently experience long-lasting sequelae but little is known about the eventual persistence of this immune alteration. Herein, we evaluated Toll-like receptor-induced cytokine responses in a cohort of mild to critical patients during acute or convalescent phases (n=97). In the acute phase, we observed impaired cytokine production by monocytes in the most severe patients. This capacity was globally restored in convalescent patients. Yet, we observed increased responsiveness to TLR1/2 ligation in patients that recovered from severe disease, indicating that these cells display distinct functional properties at the different stages of the disease. We identified a specific transcriptomic and epigenomic state in monocytes from acute severe patients that can account for their functional refractoriness. The molecular profile of monocytes from recovering patients was distinct and characterized by increased chromatin accessibility at AP1 and MAF loci. These results demonstrate that severe COVID-19 infection has a profound impact on the differentiation status and function of circulating monocytes both during the acute and the convalescent phases in a completely distinct manner. This could have important implications for our understanding of short and long-term COVID19-related morbidity.
Elisa Brauns, Abdulkader Azouz, David Grimaldi, Hanxi Xiao, Séverine Thomas, Muriel Nguyen, Véronique Olislagers, Ines Vu Duc, Carmen Orte Cano, Véronique Del Marmol, Pieter Pannus, Frédérick Libert, Sven Saussez, Nicolas Dauby, Jishnu Das, Arnaud Marchant, Stanislas Goriely
To elicit effective anti-tumor responses, CD8+ T cells need to infiltrate tumors and sustain their effector function within the immunosuppressive tumor microenvironment. Here we evaluate the role of MNK kinase activity in regulating CD8+ T cell infiltration and anti-tumor activity in pancreatic and thyroid tumors. We first show that human pancreatic and thyroid tumors with increased MNK kinase activity are associated with decreased infiltration by CD8+ T cells. We then show that while MNK inhibitors increase CD8+ T cells in these tumors, they induce a T cell exhaustion phenotype in the tumor microenvironment. Mechanistically, we show that the exhaustion phenotype is not caused by upregulation of PD-L1 but by tumor-associated macrophages (TAMs) becoming more immunosuppressive following MNK inhibitor treatment. Reversal of CD8+ T cell exhaustion by an anti-PD-1 antibody or TAM depletion synergizes with MNK inhibitors to control tumor growth and prolong animal survival. Importantly, we show in ex vivo human pancreatic tumor slice cultures that MNK inhibitors increase the expression of markers associated with immunosuppressive TAMs. Together, these findings demonstrate a previously unknown role of MNK kinases in modulating a pro-tumoral phenotype in macrophages and identify combination regimens involving MNK inhibitors to enhance anti-tumor immune responses.
Thao N.D. Pham, Christina Spaulding, Mario A. Shields, Anastasia E. Metropulos, Dhavan N. Shah, Mahmoud G. Khalafalla, Daniel R. Principe, David J. Bentrem, Hidayatullah G. Munshi
INTRODUCTION. Immune cell profiling of primary and metastatic central nervous system (CNS) tumors has been focused on the tumor, not the tumor microenvironment (TME), or have been analyzed via biopsies. METHODS. En bloc resections of glioma (n=10) and lung metastasis (n=10) underwent tissue segmentation and high dimension opal 7-color multiplex imaging. Single cell RNA analyses inferred immune cell functionality. RESULTS. Within gliomas, T cells were localized to the infiltrating edge and perivascular space of tumors, while residing mostly in the stroma of metastatic tumors. CD163+ macrophages were evident throughout the TME of metastatic tumors, whereas in gliomas, CD68+, CD11c+CD68+, and CD11c+CD68+CD163+ cell subtypes, were commonly observed. In lung metastases, T cells interact with CD163+ macrophages as dyads and clusters at the brain-tumor interface and within the tumor itself, and as clusters within the necrotic core. In contrast, gliomas typically lack dyad and cluster interactions, except for T cell-CD68+cell dyads within the tumor. Analysis of transcriptomic data in glioblastomas revealed that innate immune cells express both pro-inflammatory and immune suppressive gene signatures. CONCLUSION. Our results show that immunosuppressive macrophages are abundant within the TME, and that the immune cell interactome between cancer lineages is distinct. Further, these data provide information for evaluating the role of different immune cell populations in brain tumor growth and therapeutic responses.
Hinda Najem, Martina Ott, Cynthia Kassab, Arvind Rao, Ganesh Rao, Anantha Marisetty, Adam M. Sonabend, Craig Horbinski, Roel Verhaak, Anand Shankar, Santhoshi N. Krishnan, Frederick S. Varn, Víctor A. Arrieta, Pravesh Gupta, Sherise D. Ferguson, Jason T. Huse, Gregory N. Fuller, James P. Long, Daniel E. Winkowski, Benjamin A. Freiberg, C. David James, Leonidas C. Platanias, Maciej S. Lesniak, Jared K. Burks, Amy B. Heimberger
The persistence of virally infected cells as reservoirs despite effective antiretroviral therapy is a major barrier to HIV/simian immunodeficiency virus (SIV) cure. These reservoirs are predominately contained within cells present in the B cell follicles (BCF) of secondary lymphoid tissues, a site that is characteristically difficult for most cytolytic antiviral effector cells to penetrate. Here, we identified a population of natural killer (NK) cells in macaque lymph nodes that expressed BCF-homing receptor C-X-C chemokine receptor 5 (CXCR5) and accumulated within BCF during chronic SHIV infection. These CXCR5+ follicular NK cells exhibited an activated phenotype coupled with heightened effector functions and a unique transcriptome characterized by elevated expression of cytolytic mediators (e.g. perforin and granzymes, LAMP-1). CXCR5+ NK cells exhibited high expression of FcγRIIa and FcγRIIIa, suggesting a potential for elevated antibody-dependent effector functionality. Consistently, accumulation of CXCR5+ NK cells showed a strong inverse association with plasma viral load and the frequency of germinal center follicular helper T cells that comprises a significant fraction of the viral reservoir. Moreover, CXCR5+ NK cells showed increased expression of transcripts associated with IL-12 and IL-15 signaling compared to the CXCR5- subset. Indeed, in vitro treatment with IL-12 and IL-15 enhanced the proliferation of CXCR5+ granzyme-B+ NK cells. Our findings suggest that follicular homing NK cells might be important in immune control of chronic SHIV infection, which may have important implications for HIV cure strategies.
Sheikh Abdul Rahman, James Billinglsley, Ashish Arunkumar Sharma, Tiffany M. Styles, Sakthivel Govindaraj, Uma Shanmugasundaram, Hemalatha Babu, Susan Pereira Ribeiro, Syed A. Ali, Gregory K. Tharp, Chris Ibegbu, Stephen Waggoner, R. Paul Johnson, Rafick-Pierre Sékaly, Francois Villinger, Steven E. Bosinger, Rama Rao Amara, Vijayakumar Velu
We previously found that kidney-infiltrating T cells (KITs) in murine lupus nephritis (LN) resembled dysfunctional T cells that infiltrate tumors. This unexpected finding raised the question of how to reconcile the “exhausted” phenotype of KITs with ongoing tissue destruction in LN. To address this, we performed scRNA-seq and TCR-seq of KITs in murine lupus models. We found that CD8 KITs exist first in a transitional state, before clonally expanding and evolving toward exhaustion. On the other hand, CD4 KITs did not fit into current differentiation paradigms, but included both hypoxic and cytotoxic subsets with a pervasive exhaustion signature. Thus, autoimmune nephritis is unlike acute pathogen immunity; rather the kidney microenvironment suppresses T cells by progressively inducing exhausted states. Our findings suggest that lupus nephritis, a chronic condition, results from slow evolution of damage caused by dysfunctional T cells and their precursors on the way to exhaustion. These findings have implications for both autoimmunity and tumor immunology.
Shuchi Smita, Maria Chikina, Mark J. Shlomchik, Jeremy S. Tilstra
Therapeutic IL-12 has demonstrated the ability to reduce local immune suppression in preclinical models, but clinical development has been limited by severe inflammation-related adverse events with systemic administration. Here, we show that potent immunologic tumor control of established syngeneic carcinomas can be achieved by i.t. administration of a tumor-targeted IL-12 antibody fusion protein (NHS–rmIL-12) using sufficiently low doses to avoid systemic toxicity. Single-cell transcriptomic analysis and ex vivo functional assays of NHS–rmIL-12–treated tumors revealed reinvigoration and enhanced proliferation of exhausted CD8+ T lymphocytes, induction of Th1 immunity, and a decrease in Treg number and suppressive capacity. Similarly, myeloid cells transitioned toward inflammatory phenotypes and displayed reduced suppressive capacity. Cell type–specific IL-12 receptor–KO BM chimera studies revealed that therapeutic modulation of both lymphoid and myeloid cells is required for maximum treatment effect and tumor cure. Study of single-cell data sets from human head and neck carcinomas revealed IL-12 receptor expression patterns similar to those observed in murine tumors. These results describing the diverse mechanisms underlying tumor-directed IL-12–induced antitumor immunity provide the preclinical rationale for the clinical study of i.t. NHS–IL-12.
Youji Hong, Yvette Robbins, Xinping Yang, Wojciech K. Mydlarz, Anastasia Sowers, James B. Mitchell, James L. Gulley, Jeffrey Schlom, Sofia R. Gameiro, Cem Sievers, Clint T. Allen
Phosphopeptides derived from dysregulated protein phosphorylation in cancer cells can be processed and presented by MHC class I and class II molecules and, therefore, represent an untapped class of tumor-specific antigens that could be used as widely expressed “public” cancer neoantigens (NeoAgs). We generated a TCR mimic (TCRm) mAb, 6B1, specific for a phosphopeptide derived from insulin receptor substrate 2 (pIRS2) presented by HLA-A*02:01. The pIRS2 epitope’s presentation by HLA-A*02:01 was confirmed by mass spectrometry. The TCRm 6B1 specifically bound to pIRS2/HLA-A2 complex on tumor cell lines that expressed pIRS2 in the context of HLA-A*02:01. Bispecific mAbs engaging CD3 of T cells were able to kill tumor cell lines in a pIRS2- and HLA-A*02:01–restricted manner. Structure modeling shows a prerequisite for an arginine or lysine at the first position to bind mAb. Therefore, 6B1 could recognize phosphopeptides derived from various phosphorylated proteins with similar amino acid compositions. This raised the possibility that a TCRm specific for the pIRS2/HLA-A2 complex could target a range of phosphopeptides presented by HLA-A*02:01 in various tumor cells. This is the first TCRm mAb to our knowledge targeting a phosphopeptide/MHC class I complex; the potential of this class of agents for clinical applications warrants further investigation.
Tao Dao, Sung Soo Mun, Zaki Molvi, Tatyana Korontsvit, Martin G. Klatt, Abdul G. Khan, Elisabeth K. Nyakatura, Mary Ann Pohl, Thomas E. White, Paul J. Balderes, Ivo C. Lorenz, Richard J. O’Reilly, David A. Scheinberg
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccines pose as the most effective approach for mitigating COVID-19 pandemic. High-degree efficacy of SARS-CoV-2 vaccines in clinical trials indicates that vaccination invariably induces an adaptive immune response in vaccine recipients. However, the emergence of breakthrough infections in vaccinated individuals suggest that the breadth and magnitude vaccine-induced adaptive immune response may varies. We assessed vaccine-induced SARS-CoV-2 T-cell response in twenty-one vaccinated individuals and found that SARS-CoV-2 specific T-cells were invariably detected in all individuals. However, the magnitude and breadth of SARS-CoV-2 specific T-cell response varied. Vaccination induced mainly a CD4+ T-cell dominant SARS-CoV-2 specific immune response and the frequencies of SARS-CoV-2 specific T-cell varied across vaccinated individuals. To gain insights into whether SARS-CoV-2 vaccines can induce a long-lived T-cell immune response we investigated differentiation states and cytokine profiles to identify immune features associated with superior recall function and longevity. We identified distinct hierarchically organized differentiation states and cytokine expression patterns. SARS-CoV-2 specific CD4+ T-cells were polyfunctional and produced high levels of IL-2, which could be associated with superior longevity. Stratifying the vaccinated individuals based on the breadth and magnitude of vaccine-induced SARS-CoV-2 response identified two distinct response groups: individuals with high abundance vs low abundance of SARS-CoV-2 T-cells. The fractions of TNF-a and IL-2 producing SARS-CoV-2 T-cells were the main determinants distinguishing high vs low responders. Lastly, we identified the majority of vaccine-induced SARS-CoV-2 T-cells were reactive against conserved regions of mutant S-protein, suggesting that vaccine-induced SARS-CoV-2 T-cells could provide continued protection against emerging variants-of-concern.
Li Li, Muharrem Muftuoglu, Shaoheng Liang, Mahesh Basyal, Jiangxing LV, Mehmet E. Akdogan, Ken Chen, Michael Andreeff, Simrit Parmar
BACKGROUND. There is a need to support the diagnosis of anaphylaxis by objective markers. miRNAs are promising non-coding RNA species that may serve as serological biomarkers, but their use in diagnosing anaphylaxis has not been systematically studied. We aimed to comprehensively investigate serum biomarker profiles (proteins, lipids, and miRNAs) to support the diagnosis of anaphylaxis. METHODS. Adult patients admitted to the emergency room with a diagnosis of anaphylaxis (< 3 hours) were included. Blood samples were taken upon emergency room arrival and one month later. RESULTS. Next-generation sequencing of 18 samples (6 anaphylaxis patients in acute and non-acute condition, respectively, and 6 healthy controls) identified hsa-miR-451a to be elevated during anaphylaxis and verified by RT-qPCR in the remaining cohort. The random forest classifier enabled us to classify anaphylaxis with high accuracy using a composite model. We identified tryptase, 9α,11β-PGF2, apolipoprotein A1, and hsa-miR-451a as serological biomarkers of anaphylaxis. These predictors qualified as serological biomarkers individually but performed better in combination. CONCLUSION. Unexpectedly, hsa-miR-451a was identified as the most relevant biomarker in our data set. We were also able to distinguish between patients with a history of anaphylaxis and healthy individuals with higher accuracy than any other available model. Future studies will need to verify miRNA biomarker utility in real-life clinical settings. FUNDING. This work is funded by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) as part of the clinical research unit (CRU339): Food allergy and tolerance (FOOD@) and a grant to MW (Wo541-16-2); project numbers (409525714, 428094283, 428447634, 264921598).
Wojciech Francuzik, Kristijan Pažur, Magdalena Dalke, Sabine Dölle-Bierke, Magda Babina, Margitta Worm
The intensity and longevity of inflammatory responses to inhaled allergens is determined largely by the balance between effector and regulatory immune responses, but the mechanisms that determine the relative magnitudes of these opposing forces remain poorly understood. We have found that the type of adjuvant used during allergic sensitization has a profound effect on both the nature and longevity of the pulmonary inflammation triggered by subsequent reexposure to that same provoking allergen. TLR ligand adjuvants and house dust extracts primed immune responses characterized by a mixed neutrophilic and eosinophilic inflammation that was suppressed by multiple daily allergen challenges. During TLR ligand–mediated allergic sensitization, mice displayed transient airway neutrophilia, which triggered the release of TGF-β into the airway. This neutrophil-dependent production of TGF-β during sensitization had a delayed, suppressive effect on eosinophilic responses to subsequent allergen challenge. Neutrophil depletion during sensitization did not affect numbers of Foxp3+ Tregs but increased proportions of Gata3+CD4+ T cells, which, upon their transfer to recipient mice, triggered stronger eosinophilic inflammation. Thus, a neutrophil/TGF-β axis acts during TLR-mediated allergic sensitization to fine-tune the phenotype of developing allergen-specific CD4+ T cells and limit their pathogenicity, suggesting a novel immunotherapeutic approach to control eosinophilia in asthma.
Gregory S. Whitehead, Seddon Y. Thomas, Keiko Nakano, Derek J. Royer, Catherine G. Burke, Hideki Nakano, Donald N. Cook
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