T cell exhaustion is a state of T cell dysfunction associated with expression of programmed death 1 (PD-1). Exhausted CD8 T cells are maintained by self-renewing stem-like T (TSL) cells that provide differentiated TIM3+ cells, a part of which possesses effector-like properties. PD-1-targeted therapies enhance T cell response by promoting differentiation of TSL cells toward TIM3+ cells, but the role of mTOR during T cell exhaustion remains elusive. Here, we show that mTOR inhibition has distinct outcomes during the beginning of and after the establishment of chronic viral infection. Blocking mTOR during the T cell expansion phase enhanced the T cell response by accumulating TSL cells, leading to improved efficacy of PD-1 immunotherapy. Whereas, after exhaustion progressed, mTOR inhibition caused immunosuppression characterized by decreased TIM3+ cells and increased viral load with minimal changes in TSL cells. Mechanistically, a cell-intrinsic mTOR signal was vital for differentiation of TSL cells into the TIM3+ state in the early and late phases of chronic infection as well as during PD-1 immunotherapy. Thus, PD-1 blockade worked after cessation of mTOR inhibition but simultaneous treatment failed to induce functional TIM3+ cells, reducing efficacy of PD-1 immunotherapy. Our data demonstrate that mTOR regulates T cell exhaustion and have important implications for combination cancer therapies with PD-1 blockade
Satomi Ando, Charles Perkins, Yamato Sajiki, Chase Chastain, Rajesh M. Valanparambil, Andreas Wieland, William H. Hudson, Masao Hashimoto, Suresh S. Ramalingam, Gordon J. Freeman, Rafi Ahmed, Koichi Araki
An effective adaptive immune response depends on the organized architecture of secondary lymphoid organs, including the lymph nodes (LNs). While the cellular composition and microanatomy of LNs under steady state are well defined, the impact of chronic tissue inflammation on the structure and function of draining LNs is incompletely understood. Here we showed that Mycobacterium tuberculosis infection remodeled LN architecture by increasing the number and paracortical translocation of B cells. The formation of paracortical B lymphocyte and CD35+ follicular dendritic cell clusters dispersed CCL21-producing fibroblastic reticular cells and segregated pathogen-containing myeloid cells from antigen-specific CD4+ T cells. Depletion of B cells restored the chemokine and lymphoid structure and reduced bacterial burdens in LNs of the chronically infected mice. Importantly, this remodeling process impaired activation of naive CD4+ T cells in response to mycobacterial and unrelated antigens during chronic tuberculosis infection. Our studies reveal a mechanism in the regulation of LN microanatomy during inflammation and identify B cells as a critical element limiting the T cell response to persistent intracellular infection in LNs.
Lina Daniel, Nayan D. Bhattacharyya, Claudio Counoupas, Yi Cai, Xinchun Chen, James A. Triccas, Warwick J. Britton, Carl G. Feng
The SARS-CoV-2 spike (S) glycoprotein is synthesized as large precursor protein and must be activated by proteolytic cleavage into S1 and S2. A recombinant modified vaccinia virus Ankara (MVA) expressing native, full-length S protein (MVA-SARS-2-S) is currently under investigation as candidate vaccine in phase I clinical studies. Initial results from immunogenicity monitoring revealed induction of S-specific antibodies binding to S2, but low-level antibody responses to the S1 domain. Follow-up investigations of native S antigen synthesis in MVA-SARS-2-S infected cells revealed limited levels of S1 protein on the cell surface. In contrast, we found superior S1 cell surface presentation upon infection with a recombinant MVA expressing a stabilized version of SARS-CoV-2 S protein with an inactivated S1/2 cleavage site and K986→P and V987→P mutations (MVA-SARS-2-ST). When comparing immunogenicity of MVA vector vaccines, mice vaccinated with MVA-SARS-2-ST mounted substantial levels of S broadly reactive antibodies that effectively neutralized different SARS-CoV-2 variants. Importantly, intramuscular MVA-SARS-2-ST immunization of hamsters and mice resulted in potent immune responses upon challenge infection and protected from disease and severe lung pathology. Our results suggest that MVA-SARS-2-ST represents an improved clinical candidate vaccine and that the presence of plasma membrane-bound S1 is highly beneficial to induce protective antibody levels.
Christian Meyer zu Natrup, Alina Tscherne, Christine Dahlke, Malgorzata Ciurkiewicz, Dai-Lun Shin, Anahita Fathi, Cornelius Rohde, Georgia Kalodimou, Sandro Halwe, Leonard Limpinsel, Jan H. Schwarz, Martha Klug, Meral Esen, Nicole Schneiderhan-Marra, Alex Dulovic, Alexandra Kupke, Katrin Brosinski, Sabrina Clever, Lisa-Marie Schünemann, Georg Beythien, Federico Armando, Leonie Mayer, Leonie M. Weskamm, Sylvia Jany, Astrid Freudenstein, Tamara Tuchel, Wolfgang Baumgärtner, Peter Kremsner, Rolf Fendel, Marylyn M. Addo, Stephan Becker, Gerd Sutter, Asisa Volz
BACKGROUND Several molecular imaging strategies can identify bacterial infections in humans. PET affords the potential for sensitive infection detection deep within the body. Among PET-based approaches, antibiotic-based radiotracers, which often target key bacterial-specific enzymes, have considerable promise. One question for antibiotic radiotracers is whether antimicrobial resistance (AMR) reduces specific accumulation within bacteria, diminishing the predictive value of the diagnostic test.METHODS Using a PET radiotracer based on the antibiotic trimethoprim (TMP), [11C]-TMP, we performed in vitro uptake studies in susceptible and drug-resistant bacterial strains and whole-genome sequencing (WGS) in selected strains to identify TMP resistance mechanisms. Next, we queried the NCBI database of annotated bacterial genomes for WT and resistant dihydrofolate reductase (DHFR) genes. Finally, we initiated a first-in-human protocol of [11C]-TMP in patients infected with both TMP-sensitive and TMP-resistant organisms to demonstrate the clinical feasibility of the tool.RESULTS We observed robust [11C]-TMP uptake in our panel of TMP-sensitive and -resistant bacteria, noting relatively variable and decreased uptake in a few strains of P. aeruginosa and E. coli. WGS showed that the vast majority of clinically relevant bacteria harbor a WT copy of DHFR, targetable by [11C]-TMP, and that despite the AMR, these strains should be “imageable.” Clinical imaging of patients with [11C]-TMP demonstrated focal radiotracer uptake in areas of infectious lesions.CONCLUSION This work highlights an approach to imaging bacterial infection in patients, which could affect our understanding of bacterial pathogenesis as well as our ability to better diagnose infections and monitor response to therapy.TRIAL REGISTRATION ClinicalTrials.gov NCT03424525.FUNDING Institute for Translational Medicine and Therapeutics, Burroughs Wellcome Fund, NIH Office of the Director Early Independence Award (DP5-OD26386), and University of Pennsylvania NIH T32 Radiology Research Training Grant (5T32EB004311-12).
Iris K. Lee, Daniel A. Jacome, Joshua K. Cho, Vincent Tu, Anthony J. Young, Tiffany Dominguez, Justin D. Northrup, Jean M. Etersque, Hsiaoju S. Lee, Andrew Ruff, Ouniol Aklilu, Kyle Bittinger, Laurel J. Glaser, Daniel Dorgan, Denis Hadjiliadis, Rahul M. Kohli, Robert H. Mach, David A. Mankoff, Robert K. Doot, Mark A. Sellmyer
Invasive bacterial infections remain a major cause of human morbidity. Group B Streptococcus (GBS) are Gram-positive bacteria that cause invasive infections in humans. Here, we show that Factor XIIIA (FXIIIA) -deficient female mice exhibited significantly increased susceptibility to GBS infections. Additionally, female wild-type mice had increased levels of FXIIIA and were more resistant to GBS infection compared to isogenic male mice. We observed that administration of exogenous FXIIIA to male mice increased host resistance to GBS infection. Conversely, administration of a FXIIIA transglutaminase inhibitor to female mice decreased host resistance to GBS infection. Interestingly, male gonadectomized mice exhibited decreased sensitivity to GBS infection, suggesting a role for gonadal androgens in host susceptibility. FXIIIA promoted GBS entrapment within fibrin clots by crosslinking fibronectin with ScpB, a fibronectin binding GBS surface protein. Thus, ScpB-deficient GBS exhibited decreased entrapment within fibrin clots in vitro and increased dissemination during systemic infections. Finally, using mice where FXIIIA expression was depleted in mast cells, we observed that mast cell derived FXIIIA contributes to host defense against GBS infection. Our studies provide insights into the effect of sexual dimorphism and mast cells on FXIIIA expression and its interactions with GBS adhesins that mediate bacterial dissemination and pathogenesis.
Adrian M. Piliponsky, Kavita Sharma, Phoenicia Quach, Alyssa Brokaw, Shayla Nguyen, Austyn Orvis, Siddhartha S. Saha, Nyssa Becker Samanas, Ravin Seepersaud, Yu Ping Tang, Emily Mackey, Gauri Bhise, Claire Gendrin, Anna Furuta, Albert J. Seo, Eric Guga, Irina Miralda, Michelle M. Coleman, Erin L. Sweeney, Charlotte A. Bäuml, Diana Imhof, Jessica M. Snyder, Adam J. Moeser, Lakshmi Rajagopal
Once-weekly oral dose of isoniazid and rifapentine for 12 weeks (3HP) is recommended by CDC for treatment of latent tuberculosis infection (LTBI). The aim of this study is to assess 3HP-mediated clearance of Mtb bacteria in macaques with asymptomatic LTBI. Twelve Indian rhesus macaques were infected with low dose (~10 CFU) of Mtb CDC1551 via aerosol. Six animals were treated with 3HP and six were left untreated. The animals were imaged via positron emissions tomography – computed tomography (PET/CT) at frequent intervals. Upon treatment completion, all animals except one were coinfected with simian immunodeficiency virus to assess reactivation of LTBI to active TB disease. Four of six treated macaques showed no evidence of persistent bacilli or extrapulmonary spread until study end-point. PET/CT demonstrated the presence of significantly more granulomas in untreated animals relative to the treated group. The untreated animals harbored persistent bacilli and demonstrated TB reactivation following SIV coinfection while none of the treated animals reactivated to active TB disease (ATB). 3HP treatment effectively reduced persistent infection with Mtb and prevented reactivation of TB disease in latently infected macaques.
Riti Sharan, Shashank R. Ganatra, Dhiraj K. Singh, Journey Cole, Taylor W. Foreman, Rajesh Thippeshappa, Charles A. Peloquin, Vinay Shivanna, Olga Gonzalez, Cheryl L. Day, Neel R. Gandhi, Edward J. Dick Jr., Shannan Hall-Ursone, Smriti Mehra, Larry S. Schlesinger, Jyothi Rengarajan, Deepak Kaushal
The focus of hepatitis B functional cure, defined as sustained loss of hepatitis B surface antigen (HBsAg) and HBV DNA from blood, is on eliminating or silencing the intranuclear template for HBV replication, covalently closed circular DNA (cccDNA). However, HBsAg also derives from HBV DNA integrated into the host genome (iDNA). Little is known about the contribution of iDNA to circulating HBsAg with current therapeutics. We applied a multiplex ddPCR assay to demonstrate that iDNA is responsible for maintaining HBsAg quantities in some individuals. Using paired bulk liver tissue from 16 HIV/HBV coinfected persons on nucleos(t)ide analogue (NUC) therapy, we demonstrate that people with larger HBsAg declines between biopsies derive HBsAg from cccDNA whereas people with stable HBsAg levels derive predominantly from iDNA. We applied our assay to individual hepatocytes in paired tissues from three people and demonstrated that the individual with significant HBsAg decline had a commensurate loss of infected cells with transcriptionally active cccDNA, while individuals without HBsAg decline had stable or increasing numbers of cells producing HBsAg from iDNA. We demonstrate that while NUC therapy may be effective at controlling cccDNA replication and transcription, innovative treatments are required to address iDNA transcription that sustains HBsAg production.
Tanner Grudda, Hyon S. Hwang, Maraake Taddese, Jeffrey Quinn, Mark S. Sulkowski, Richard K. Sterling, Ashwin Balagopal, Chloe L. Thio
Mitohormesis defines the increase in fitness mediated by adaptive responses to mild mitochondrial stress. Tetracyclines inhibit not only bacterial but also mitochondrial translation, thus imposing a low level of mitochondrial stress to eukaryotic cells. We demonstrate in cell and germ-free mouse models, that tetracyclines induce a mild adaptive mitochondrial stress response (MSR), involving both the ATF4-mediated integrative stress response and type I interferon (IFN) signaling. To overcome the interferences of tetracyclines with the host microbiome, we identify tetracycline derivatives that have minimal antimicrobial activity, yet retain full capacity to induce the MSR, such as the lead compound, 9-tert-butyldoxycycline (9-TB). The MSR induced by Doxycycline (Dox) and 9-TB improves survival and disease tolerance against lethal influenza virus (IFV) infection when given preventively. 9-TB, unlike Dox, did not affect the gut microbiome and showed also encouraging results against IFV when given in a therapeutic setting. Tolerance to IFV infection is associated with the induction of genes involved in lung epithelial cell and cilia function, and with down-regulation of inflammatory and immune gene sets in lungs, liver, and kidneys. Mitohormesis induced by non-antimicrobial tetracyclines and the ensuing IFN response may dampen excessive inflammation and tissue damage during viral infections, opening innovative therapeutic avenues.
Adrienne Mottis, Terytty Y. Li, Gaby El Alam, Alexis Rapin, Elena Katsyuba, David Liaskos, Davide D'Amico, Nicola L. Harris, Mark C. Grier, Laurent Mouchiroud, Mark L. Nelson, Johan Auwerx
Human cytomegalovirus (HCMV) is the most common congenital infection and a leading cause of stillbirth, neurodevelopmental impairment, and pediatric hearing loss worldwide. Development of a maternal vaccine or therapeutic to prevent congenital HCMV has been hindered by limited knowledge of the immune responses that protect against HCMV transmission in utero. To identify protective antibody responses, we measured HCMV-specific IgG binding and anti-viral functions in paired maternal and cord blood sera from HCMV seropositive transmitting (n=41) and non-transmitting (n=40) mother-infant dyads identified via a large U.S.-based public cord blood bank. We found that high avidity IgG binding to HCMV and antibody-dependent cellular phagocytosis (ADCP) were associated with reduced risk of congenital HCMV infection. We also determined that HCMV-specific IgG activation of FcγRI and FcγRII was enhanced in non-transmitting dyads and that increased ADCP responses were mediated through both FcγRI and FcγRIIA expressed on human monocytes. These findings suggest that engagement of FcγRI/FcγRIIA and Fc effector functions including ADCP may protect against congenital HCMV infection. Taken together, these data can guide future prospective studies on immune correlates against cCMV transmission and inform HCMV vaccine and immunotherapeutic development.
Eleanor C. Semmes, Itzayana G. Miller, Courtney E. Wimberly, Caroline T. Phan, Jennifer A. Jenks, Melissa J. Harnois, Stella J. Berendam, Helen Webster, Jillian H. Hurst, Joanne Kurtzberg, Genevieve G. Fouda, Kyle M. Walsh, Sallie R. Permar
Plasmodium falciparum (P. falciparum) induces trained innate immune responses in vitro, where initial stimulation of adherent PBMCs with P. falciparum–infected RBCs (iRBCs) results in hyperresponsiveness to subsequent ligation of TLR2. This response correlates with the presence of T and B lymphocytes in adherent PBMCs, suggesting that innate immune training is partially due to adaptive immunity. We found that T cell–depleted PBMCs and purified monocytes alone did not elicit hyperproduction of IL-6 and TNF-α under training conditions. Analysis of P. falciparum–trained PBMCs showed that DCs did not develop under control conditions, and IL-6 and TNF-α were primarily produced by monocytes and DCs. Transwell experiments isolating purified monocytes from either PBMCs or purified CD4+ T cells, but allowing diffusion of secreted proteins, enabled monocytes trained with iRBCs to hyperproduce IL-6 and TNF-α after TLR restimulation. Purified monocytes stimulated with IFN-γ hyperproduced IL-6 and TNF-α, whereas blockade of IFN-γ in P. falciparum–trained PBMCs inhibited trained responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from patients with malaria showed persistently open chromatin at genes that appeared to be trained in vitro. Together, these findings indicate that the trained immune response of monocytes to P. falciparum is not completely cell intrinsic but depends on soluble signals from lymphocytes.
Juliet N. Crabtree, Daniel R. Caffrey, Leandro de Souza Silva, Evelyn A. Kurt-Jones, Katherine Dobbs, Arlene Dent, Katherine A. Fitzgerald, Douglas T. Golenbock
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