Antiretroviral therapy duration and immunometabolic state determine efficacy of ex vivo dendritic cell-based treatment restoring functional HIV-specific CD8+ T cells in people living with HIV

HIV; Immunotherapy; MetabolismVIH; Inmunoterapia; MetabolismoVIH; Immunoteràpia; MetabolismeBackground Dysfunction of CD8+ T cells in people living with HIV-1 (PLWH) receiving anti-retroviral therapy (ART) has restricted the efficacy of dendritic cell (DC)-based immunotherapies against HIV-1. Heterogeneous immune exhaustion and metabolic states of CD8+ T cells might differentially associate with dysfunction. However, specific parameters associated to functional restoration of CD8+ T cells after DC treatment have not been investigated. Methods We studied association of restoration of functional HIV-1-specific CD8+ T cell responses after stimulation with Gag-adjuvant-primed DC with ART duration, exhaustion, metabolic and memory cell subsets profiles. Findings HIV-1-specific CD8+ T cell responses from a larger proportion of PLWH on long-term ART (more than 10 years; LT-ARTp) improved polyfunctionality and capacity to eliminate autologous p24+ infected CD4+ T cells in vitro. In contrast, functional improvement of CD8+ T cells from PLWH on short-term ART (less than a decade; ST-ARTp) after DC treatment was limited. This was associated with lower frequencies of central memory CD8+ T cells, increased co-expression of PD1 and TIGIT and reduced mitochondrial respiration and glycolysis induction upon TCR activation. In contrast, CD8+ T cells from LT-ARTp showed increased frequencies of TIM3+ PD1− cells and preserved induction of glycolysis. Treatment of dysfunctional CD8+ T cells from ST-ARTp with combined anti-PD1 and anti-TIGIT antibodies plus a glycolysis promoting drug restored their ability to eliminate infected CD4+ T cells. Interpretation Together, our study identifies specific immunometabolic parameters for different PLWH subgroups potentially useful for future personalized DC-based HIV-1 vaccines.EMG was supported by the NIH R21 program (R21AI140930), the Ramón y Cajal Program (RYC2018-024374-I), the MINECO/FEDER RETOS program (RTI2018-097485-A-I00), by Comunidad de Madrid Talento Program (2017-T1/BMD-5396) and by Gilead becas de investigación (GLD19/00168). EMG and IDS are supported by Centro de Investigación Biomédica en Red (CIBERINF) de Enfermedades Infecciosas (CB21/13/00107). MCM was supported by NIH R21 program (R21AI140930), “La Caixa Banking Foundation (H20-00218) and Gilead becas de investigación (GLD19/00168). MJB is supported by the Miguel Servet program funded by the Spanish Health Institute Carlos III (CP17/00179), the MINECO/FEDER RETOS program (RTI2018-101082-B-100), and Fundació La Marató TV3 (201805-10FMTV3). EMG and MJB are both funded by “La Caixa Banking Foundation (H20-00218) and by REDINCOV grant from Fundació La Marató TV3. FSM was supported by SAF2017-82886-R and PDI-2020-120412RB-I00 grants from the Ministerio de Ciencia e Innovación, and HR17-00016 grant from “La Caixa Banking Foundation. HF was funded by PI21/01583 grant from Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III. MJC was supported by PID2019-104406RB-I00 from Ministerio de Ciencia e Innovación. ISC was funded by the CM21/00157 Rio-Hortega grant. IT was supported by grant for the promotion of research studies master-UAM 2021

Aurora A drives early signalling and vesicle dynamics during T-cell activation

Aurora A is a serine/threonine kinase that contributes to the progression of mitosis by inducing microtubule nucleation. Here we have identified an unexpected role for Aurora A kinase in antigen-driven T-cell activation. We find that Aurora A is phosphorylated at the immunological synapse (IS) during TCR-driven cell contact. Inhibition of Aurora A with pharmacological agents or genetic deletion in human or mouse T cells severely disrupts the dynamics of microtubules and CD3z-bearing vesicles at the IS. The absence of Aurora A activity also impairs the activation of early signalling molecules downstream of the TCR and the expression of IL-2, CD25 and CD69. Aurora A inhibition causes delocalized clustering of Lck at the IS and decreases phosphorylation levels of tyrosine kinase Lck, thus indicating Aurora A is required for maintaining Lck active. These findings implicate Aurora A in the propagation of the TCR activation signal.We thank S. Bartlett for English editing and critical reading of the manuscript, Dr A. Akhmanova for providing reagents, Maria Navarro for the her critical reading of the manuscript and scientific recommendations, Miguel Vicente-Manzanares for his critical reading of the manuscript, and Aitana Sanguino and Maria Jose Lopez for the technical support. We also thank the Confocal Microscopy \& Dynamic Imaging Unit (CNIC), Madrid, Spain. This study was supported by grants SAF2011-25834, SAF2014-55579-R and BIO2012-37926 from the Spanish Ministry of Economy and Competitiveness, INDISNET-S2011/BMD-2332 from the Comunidad de Madrid ERC-2011-AdG 294340-GENTRIS and ERC-2013-AdG 334763-NOVARIPP. Red Cardiovascular RD 12-0042-0056 from Instituto Salud Carlos III (ISCIII). The Centro Nacional de Investigaciones Cardiovasculares (CNIC, Spain) is supported by the Spanish Ministry of Science and Innovation, and the Pro-CNIC Foundation.S

Folding for the Immune Synapse: CCT Chaperonin and the Cytoskeleton.

Lymphocytes rearrange their shape, membrane receptors and organelles during cognate contacts with antigen-presenting cells (APCs). Activation of T cells by APCs through pMHC-TCR/CD3 interaction (peptide-major histocompatibility complex-T cell receptor/CD3 complexes) involves different steps that lead to the reorganization of the cytoskeleton and organelles and, eventually, activation of nuclear factors allowing transcription and ultimately, replication and cell division. Both the positioning of the lymphocyte centrosome in close proximity to the APC and the nucleation of a dense microtubule network beneath the plasma membrane from the centrosome support the T cell's intracellular polarity. Signaling from the TCR is facilitated by this traffic, which constitutes an important pathway for regulation of T cell activation. The coordinated enrichment upon T cell stimulation of the chaperonin CCT (chaperonin-containing tailless complex polypeptide 1; also termed TRiC) and tubulins at the centrosome area support polarized tubulin polymerization and T cell activation. The proteasome is also enriched in the centrosome of activated T cells, providing a mechanism to balance local protein synthesis and degradation. CCT assists the folding of proteins coming from de novo synthesis, therefore favoring mRNA translation. The functional role of this chaperonin in regulating cytoskeletal composition and dynamics at the immune synapse is discussed.This work was supported bun the ALBA Synchrotron standard proposals 2015021148 and 2016021638 to NM-C and JMV. This study was supported by grants SAF2017-82886-R (to FS-M) and PID2019-105872GB-I00/AEI/10.13039/501100011033 (AEI/FEDER, UE) (to JMV) from the Spanish Ministry of Economy and Competitiveness (MINECO), grants INFLAMUNE-S2017/BMD-23671 (to FS-M) and P2018/NMT-4389 (to JMV) from the Comunidad de Madrid, ERC-2011-AdG 294340-GENTRIS (to FS-M), a 2019 grant from the Ramón Areces Foundation “Ciencias de la Vida y la Salud” and a 2018 grant from Ayudas Fundación BBVA a Equipos de Investigación Científica (to FS-M) and grants PRB3 (IPT17/0019—ISCIII-SGEFI/ERDF), and “La Caixa” Banking Foundation (HR17-00016 to FS-M). CIBER Cardiovascular (Fondo de Investigación Sanitaria del Instituto de Salud Carlos III and co-funding by Fondo Europeo de Desarrollo Regional FEDER). The Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC Foundation. The Centro Nacional de Biotecnología (CNB) was a Severo Ochoa Center of Excellence (MINECO award SEV 2017-0712). Funding agencies have not intervened in the design of the studies, with no copyright over the study.S

HDAC6 is a Regulator of CTL Function through Control of Lytic Granule Dynamics

Viral infections involve specific stress exposure that can influence the quality and average lifespan of an organism. The immune system acts through virus clearance from the organism. Many aspects of immune cells accounting for this response are still under study. Here, we review recent aspects of the molecular mechanisms involved in the delivery of the lethal hit by Cytotoxic T lymphocytes.This study was supported by grants SAF2014-55579-R from the Spanish Ministry of Economy and Competitiveness, INDISNETS2011/BMD-2332 from the Comunidad de Madrid (Fondos FEDER), ERC-2011-AdG 294340-GENTRIS from EU. Red Cardiovascular RD 12-0042-0056 from Instituto Salud Carlos III (ISCIII). The Centro Nacional de Investigaciones Cardiovasculares (CNIC, Spain) is supported by the Spanish Ministry of Science and Innovation and the Pro-CNIC Foundation.S

Sumoylated hnRNPA2B1 controls the sorting of miRNAs into exosomes through binding to specific motifs

Exosomes are released by most cells to the extracellular environment and are involved in cell-to-cell communication. Exosomes contain specific repertoires of mRNAs, microRNAs (miRNAs) and other non-coding RNAs that can be functionally transferred to recipient cells. However, the mechanisms that control the specific loading of RNA species into exosomes remain unknown. Here we describe sequence motifs present in miRNAs that control their localization into exosomes. The protein heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) specifically binds exosomal miRNAs through the recognition of these motifs and controls their loading into exosomes. Moreover, hnRNPA2B1 in exosomes is sumoylated, and sumoylation controls the binding of hnRNPA2B1 to miRNAs. The loading of miRNAs into exosomes can be modulated by mutagenesis of the identified motifs or changes in hnRNPA2B1 expression levels. These findings identify hnRNPA2B1 as a key player in miRNA sorting into exosomes and provide potential tools for the packaging of selected regulatory RNAs into exosomes and their use in biomedical applications.This work was supported by grants SAF2011-25834, ERC-2011-AdG, BIO2009_07990, BIO2010-17527, COST-Action BM1202, P210/BMD-2305 and Cardiovascular Network RD12-0042-0056 from Instituto Salud Carlos III. C.V.B. was supported by FPU program (Spanish Ministry of Education). M.M. was supported by the Instituto de Salud Carlos III, C.G.-V. by the Comunidad de Madrid and D.J.M.H. by Programa La Caixa.S

Priming of dendritic cells by DNA-containing extracellular vesicles from activated T cells through antigen-driven contacts

Interaction of T cell with antigen-bearing dendritic cells (DC) results in T cell activation, but whether this interaction has physiological consequences on DC function is largely unexplored. Here we show that when antigen-bearing DCs contact T cells, DCs initiate antipathogenic programs. Signals of this interaction are transmitted from the T cell to the DC, through extracellular vesicles (EV) that contain genomic and mitochondrial DNA, to induce antiviral responses via the cGAS/STING cytosolic DNA-sensing pathway and expression of IRF3-dependent interferon regulated genes. Moreover, EV-treated DCs are more resistant to subsequent viral infections. In summary, our results show that T cells prime DCs through the transfer of exosomal DNA, supporting a specific role for antigen-dependent contacts in conferring protection to DCs against pathogen infection. The reciprocal communication between innate and adaptive immune cells thus allow efficacious responses to unknown threats.We thank Dr. S. Bartlett for assistance with English editing and Dr A. Garcia-Sastre for providing reagents. This study was supported by grants SAF2017/82886-R from the Spanish Ministry of Economy and Competitiveness, CAM S2017/BMD-3671 from the Comunidad de Madrid, CIBER Cardiovascular (Fondo de Investigacion Sanitaria del Instituto de Salud Carlos III and co-funding by Fondo Europeo de Desarrollo Regional FEDER), ERC-2011-AdG 294340-GENTRIS and COST-Action BM1202 to F.S.-M.; grant SAF2015-65633-R from the Spanish Ministry of Economy and Competitiveness to J.A.E. M.M. is supported by MS14/00219 from Instituto de Salud Carlos III. Centro Nacional de Investigaciones Cardiovasculares (CNIC) is supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and the Pro-CNIC Foundation, and is a Severo Ochoa Center of Excellence (MINECO award SEV-2015-0505).S

eNOS S-nitrosylates beta-actin on Cys374 and regulates PKC-theta at the immune synapse by impairing actin binding to profilin-1

The actin cytoskeleton coordinates the organization of signaling microclusters at the immune synapse (IS); however, the mechanisms involved remain poorly understood. We show here that nitric oxide (NO) generated by endothelial nitric oxide synthase (eNOS) controls the coalescence of protein kinase C-θ (PKC-θ) at the central supramolecular activation cluster (c-SMAC) of the IS. eNOS translocated with the Golgi to the IS and partially colocalized with F-actin around the c-SMAC. This resulted in reduced actin polymerization and centripetal retrograde flow of β-actin and PKC-θ from the lamellipodium-like distal (d)-SMAC, promoting PKC-θ activation. Furthermore, eNOS-derived NO S-nitrosylated β-actin on Cys374 and impaired actin binding to profilin-1 (PFN1), as confirmed with the transnitrosylating agent S-nitroso-L-cysteine (Cys-NO). The importance of NO and the formation of PFN1-actin complexes on the regulation of PKC-θ was corroborated by overexpression of PFN1- and actin-binding defective mutants of β-actin (C374S) and PFN1 (H119E), respectively, which reduced the coalescence of PKC-θ at the c-SMAC. These findings unveil a novel NO-dependent mechanism by which the actin cytoskeleton controls the organization and activation of signaling microclusters at the IS.status: publishe

Leukemic cell-secreted interleukin-9 suppresses cytotoxic T cell-mediated killing in chronic lymphocytic leukemia

Abstract The tumor microenvironment (TME) plays a central role in the pathogenesis of chronic lymphocytic leukemia (CLL), contributing to disease progression and chemoresistance. Leukemic cells shape the TME into a pro-survival and immunosuppressive niche through contact-dependent and contact-independent interactions with the cellular components of the TME. Immune synapse (IS) formation is defective in CLL. Here we asked whether soluble factors released by CLL cells contribute to their protection from cytotoxic T cell (CTL)-mediated killing by interfering with this process. We found that healthy CTLs cultured in media conditioned by leukemic cells from CLL patients or Eμ-TCL1 mice upregulate the exhaustion marker PD-1 and become unable to form functional ISs and kill target cells. These defects were more pronounced when media were conditioned by leukemic cells lacking p66Shc, a proapoptotic adapter whose deficiency has been implicated in disease aggressiveness both in CLL and in the Eμ-TCL1 mouse model. Multiplex ELISA assays showed that leukemic cells from Eμ-TCL1 mice secrete abnormally elevated amounts of CCL22, CCL24, IL-9 and IL-10, which are further upregulated in the absence of p66Shc. Among these, IL-9 and IL-10 were also overexpressed in leukemic cells from CLL patients, where they inversely correlated with residual p66Shc. Using neutralizing antibodies or the recombinant cytokines we show that IL-9, but not IL-10, mediates both the enhancement in PD-1 expression and the suppression of effector functions in healthy CTLs. Our results demonstrate that IL-9 secreted by leukemic cells negatively modulates the anti-tumor immune abilities of CTLs, highlighting a new suppressive mechanism and a novel potential therapeutical target in CLL

The mitochondrial fission factor dynamin-related protein 1 modulates T-cell receptor signalling at the immune synapse

During antigen presentation, T cells relocalize organelles, adhesion and signalling components to the immunological synapse (IS). This study shows that the localization of mitochondria to the IS is regulated by the mitochondrial fission factor Drp1 and that this is important for T-cell activation