Biology subjects

Cicin-Sain, L.

Publications and source records attributed to Cicin-Sain, L..

At least 19 recordsLinked to original sources

bioRxiv2023

DNA methylation profiling identifies TBKBP1 as potent amplifier of cytotoxic activity in CMV-specific human CD8+ T cells

Epigenetic mechanisms stabilize gene expression patterns during CD8+ T cell differentiation. However, although adoptive transfer of virus-specific T cells is clinically applied to reduce the risk of virus infection or reactivation in immunocompromised individuals, the DNA methylation pattern of virus-specific CD8+ T cells is largely unknown. Hence, we here performed whole-genome bisulfite sequencing of cytomegalovirus-specific human CD8+ T cells and found that they display a unique DNA methylation pattern consisting of 79 differentially methylated regions when compared to bulk memory CD8+ T cells. Among them was TBKBP1, coding for TBK-binding protein 1 that can interact with TANK-binding kinase 1 (TBK1) and mediate pro-inflammatory responses in innate immune cells downstream of intracellular virus sensing. Since TBKBP1 has not yet been reported in T cells, we aimed to unravel its role in virus-specific CD8+ T cells. TBKBP1 demethylation in terminal effector CD8+ T cells correlated with TBKBP1 expression and was stable upon long-term in vitro culture. TBKBP1 overexpression resulted in enhanced TBK1 phosphorylation upon stimulation of CD8+ T cells and significantly improved their virus neutralization capacity. Collectively, our data demonstrate that TBKBP1 modulates virus-specific CD8+ T cell responses and could be exploited as therapeutic target to improve adoptive T cell therapies.

immunology↗

bioRxiv2023

Dynamic monitoring of viral gene expression reveals rapid antiviral effects of CD8 T cells recognizing the HCMV-pp65 antigen

Human Cytomegalovirus (HCMV) is a betaherpesvirus that causes severe disease in immunocompromised transplant recipients. Immunotherapy with CD8 T cells specific for HCMV antigens presented on HLA class-I molecules is explored as strategy for long-term relief to such patients, but the antiviral effectiveness of T cell preparations cannot be efficiently predicted by available methods. Therefore, we developed an Assay for Rapid Measurement of Antiviral T-cell Activity (ARMATA) by real-time automated fluorescent microscopy and used it to study the ability of CD8 T cells to neutralize HCMV and control its spread. As a proof of principle, we used TCR-transgenic T cells specific for the immunodominant HLA-A02-restricted tegumental phosphoprotein pp65. pp65 expression follows an early/late kinetic, but it is not clear at which stage of the virus cycle it acts as an antigen. We measured control of HCMV infection by T cells as early as 6 hours post infection (hpi). The timing of the antigen recognition indicated that it occurred before the late phase of the virus cycle, but also that virion-associated pp65 was not recognized during virus entry into cells. Monitoring of pp65 gene expression dynamics by reporter fluorescent genes revealed that pp65 was detectable as early as 6 hpi, and that a second and much larger bout of expression occurs in the late phase of the virus cycle by 48 hpi. Since transgenic (Tg)-pp65 specific CD8 T cells were activated even when DNA replication was blocked, our data argue that pp65 acts as an early virus gene for immunological purposes. Therefore, ARMATA does not only allow same-day identification of antiviral T-cell activity, but also provides a method to define the timing of antigen recognition in the context of HCMV infection.

immunology↗

bioRxiv2023

NFAT signaling is indispensable for persistent memory responses of MCMV-specific CD8+ T cells

Cytomegalovirus (CMV) induces a unique T-cell response, where antigen-specific populations do not contract, but rather inflate during viral latency. It has been proposed that subclinical episodes of virus reactivation feed the inflation of CMV-specific memory cells by intermittently engaging T-cell receptors (TCRs), but evidence of TCR engagement has remained lacking. Nuclear factor of activated T cells (NFAT) is a family of transcription factors, where NFATc1 and NFATc2 signal downstream of TCR in mature T lymphocytes. We show selective impacts of NFATc1 and/or NFATc2 genetic ablations on the long-term inflation of MCMV-specific CD8 T-cell responses despite largely maintained responses to acute infection. NFATc1 ablation elicited robust phenotypes in isolation, but the strongest effects were observed when both NFAT genes were missing. CMV control was impaired only when both NFATs were deleted in CD8 T cells used in adoptive immunotherapy of immunodeficient mice. Transcriptome analyses revealed that T-cell intrinsic NFAT is not necessary for CD8 T-cell priming, but rather for their maturation towards effector-memory and in particular the effector cells, which dominate the pool of inflationary cells.

immunology↗

bioRxiv2022

TMPRSS2 is essential for SARS-CoV-2 Beta and Omicron infection

The COVID-19 pandemic remains a global health threat and novel antiviral strategies are urgently needed. SARS-CoV-2 employs the cellular serine protease TMPRSS2 for entry into lung cells and TMPRSS2 inhibitors are being developed for COVID-19 therapy. However, the SARS-CoV-2 Omicron variant, which currently dominates the pandemic, prefers the endo/lysosomal cysteine protease cathepsin L over TMPRSS2 for cell entry, raising doubts whether TMPRSS2 inhibitors would be suitable for treatment of patients infected with the Omicron variant. Nevertheless, the contribution of TMPRSS2 to spread of SARS-CoV-2 in the infected host is largely unclear. Here, we show that loss of TMPRSS2 strongly reduced the replication of the Beta variant in nose, trachea and lung of C57BL mice and protected the animals from weight loss and disease. Infection of mice with the Omicron variant did not cause disease, as expected, but again TMPRSS2 was essential for efficient viral spread in the upper and lower respiratory tract. These results identify a key role of TMPRSS2 in SARS-CoV-2 Beta and Omicron infection and highlight TMPRSS2 as an attractive target for antiviral intervention.

immunology↗

bioRxiv2022

Long-term immune protection against SARS-CoV-2 escape variants upon a single vaccination with murine cytomegalovirus expressing the spike protein

Current vaccines against COVID-19 elicit immune responses that are overall strong but wane rapidly. As a consequence, the necessary booster shots have led to vaccine fatigue. Hence, vaccines that would provide lasting protection against COVID-19 are needed, but are still unavailable. Cytomegaloviruses (CMV) elicit lasting and uniquely strong immune responses. Used as vaccine vectors, they may be attractive tools that obviate the need for boosters. Therefore, we tested the murine CMV (MCMV) as a vaccine vector against COVID-19 in relevant preclinical models of immunization and challenge. We have previously developed a recombinant murine CMV (MCMV) vaccine vector expressing the spike protein of the ancestral SARS-CoV-2 (MCMVS). In this study, we show that the MCMVS elicits a robust and lasting protection in young and aged mice. Notably, S-specific humoral and cellular immunity was not only maintained but even increased over a period of at least 6 months. During that time, antibody avidity continuously increased and expanded in breadth, resulting in neutralization of genetically distant variants, like Omicron BA.1. A single dose of MCMVS conferred rapid virus clearance upon challenge. Moreover, MCMVS vaccination controlled two immune-evading variants of concern (VoCs), the Beta (B.1.135) and the Omicron (BA.1) variants. Thus, CMV vectors provide unique advantages over other vaccine technologies, eliciting broadly reactive and long-lasting immune responses against COVID-19. Authors SummaryWhile widespread vaccination has substantially reduced risks of severe COVID presentations and morbidity, immune waning and continuous immune escape of novel SARS-CoV-2 variants have resulted in a need for numerous vaccine boosters and a continuous adaptation of vaccines to new SARS-CoV-2 variants. We show in proof of principle experiments with a recombinant murine cytomegalovirus expressing the SARS-CoV-2 spike protein (MCMVS) that one immunization with a CMV vaccine vector drives enduring protection in both young and aged mice, with long-term maturation of immune responses that broaden the antiviral effects over time. Hence, this approach resolves issues of immune waning and mitigates the effects of COVID-19 evolution and immune escape, reducing the need for additional immunizations and potentially improving vaccine compliance.

immunology↗

bioRxiv2022

Pharmacological modulators of epithelial immunity uncovered by synthetic genetic tracing of SARS-CoV-2 infection responses

Epithelial immune responses govern tissue homeostasis and offer drug targets against maladaptation. Here, we report a framework to generate drug discovery-ready reporters of cellular responses to viral infection. We reverse engineered epithelial cell responses to SARS-CoV-2, the viral agent fueling the ongoing COVID-19 pandemic and designed synthetic transcriptional reporters whose molecular logic comprises interferon-/{beta}/{gamma}-, and NF-{kappa}B pathways. Such regulatory potential reflected single-cell data from experimental models to severe COVID-19 patient epithelial cells infected by SARS-CoV-2. SARS-CoV-2, type-I interferons, and RIG-I drive reporter activation. Live-cell-image-based phenotypic drug screens identified JAK inhibitors and DNA damage inducers as antagonistic modulators of epithelial cell response to interferons, RIG-I stimulation, and SARS-CoV-2. Synergistic or antagonistic modulation of the reporter by drugs underscored their similar mechanism of action. Thus, this study describes a tool for dissecting antiviral responses to infection and sterile cues, and a rapid approach to other emerging viruses of public health concern in order to discover rational drug combinations.

bioengineering↗

bioRxiv2022

RIG-I activation primes and trains innate antiviral immune memory

Adaptive processes of the innate immune system, known as trained immunity (TI), are critical to human health and disease, yet they have not been systematically investigated downstream of antiviral sensing. Here, we elucidate the potential of the antiviral cytosolic RNA receptor retinoic acid-inducible gene I (RIG-I) to train, prime and tolerize the innate immune system. Using a specific RIG-I agonist, we observed that repetitive stimulation enhanced interferon-stimulated gene (ISG) and pro-inflammatory cytokine induction in human primary monocytes, epithelial cells and fibroblasts and afforded non-specific antiviral protection. RNA sequencing revealed broad, cell type-specific transcriptional changes, indicative of priming of ISGs and training of the NF{kappa}B pathway, without measurable tolerization, while ATAC sequencing in monocytes demonstrated chromatin remodeling and enhanced accessibility of key transcription factor-binding motifs such as STAT1. Moreover, while STAT1 signaling was critically required, it was not sufficient to recapitulate RIG-I induced TI. Altogether, our data demonstrate that RIG-I-mediated TI promotes an immunologically alert state with important implications for host defense and the application of RIG-I ligands in anti-infective and anti-tumoral therapies. One Sentence SummaryRIG-I activation trains and primes innate immune response at the cellular level, affording non-specific immune protection by immune and non-immune cells.

immunology↗

bioRxiv2022

PDGFRα+ fibroblasts are a major site of cytomegalovirus replication in vivo and genome maintenance during latency

To date, no herpesvirus has been shown to latently persist in fibroblastic cells. Here, we demonstrate that mouse CMV (MCMV), a {beta}-herpesvirus, persists for the long term and across organs in PDGFR+ fibroblastic cells, with similar or higher genome loads than in the previously known sites of MCMV latency. Whereas MCMV gene transcription in PDGFR+ fibroblastic cells was almost completely silenced at 5 months post-infection, these cells gave rise to reactivated virus ex vivo, arguing that they supported latent MCMV infection. Notably, PDGFR+ fibroblastic cells also supported productive virus replication during primary MCMV infection. Mechanistically, Stat1-deficiency resulted in increased lytic but abolished latent infection of fibroblastic cells in vivo. In sum, fibroblastic cells have a dual role as a site of lytic MCMV replication and a reservoir of latent MCMV in vivo and Stat1 is critically involved in the regulation of MCMV latency.

microbiology↗

bioRxiv2022

Adaptation to ER-stress via serine glycine metabolism licences STING signalling and CMV control in intestinal epithelial cells

Inflammatory bowel diseases (IBD) are characterized by chronic relapsing inflammation of the gastrointestinal tract. While the molecular causality between endoplasmic reticulum (ER) stress and intestinal inflammation is widely accepted, the metabolic consequences of chronic ER-stress on the pathophysiology of IBD remain unclear. By using in vitro, ex vivo, in vivo mouse models and patient datasets, we identified a distinct polarisation of the mitochondrial one-carbon (1C) metabolism and a fine-tuning of the amino acid uptake in intestinal epithelial cells tailored to support GSH and NADPH metabolism upon chronic ER-stress. This metabolic phenotype strongly correlates with IBD severity and therapy-response. Mechanistically, we uncover that both chronic ER-stress and serine limitation disrupt cGAS/STING-signalling, impairing the epithelial response against viral and bacterial infection, fuelling experimental enteritis. Consequently, antioxidant treatment restores STING function and virus control. Collectively, our data highlight the importance of the plasticity of serine metabolism to allow proper cGAS/STING-signalling and innate immune responses upon chronic inflammation in the gut.

cell biology↗

bioRxiv2021

A third vaccination with a single T cell epitope protects against SARS-CoV-2 infection in the absence of neutralizing antibodies

Understanding the mechanisms and impact of booster vaccinations can facilitate decisions on vaccination programmes. This study shows that three doses of the same synthetic peptide vaccine eliciting an exclusive CD8+ T cell response against one SARS-CoV-2 Spike epitope protected all mice against lethal SARS-CoV-2 infection in the K18-hACE2 transgenic mouse model in the absence of neutralizing antibodies, while only a second vaccination with this T cell vaccine was insufficient to provide protection. The third vaccine dose of the single T cell epitope peptide resulted in superior generation of effector-memory T cells in the circulation and tissue-resident memory T (TRM) cells, and these tertiary vaccine-specific CD8+ T cells were characterized by enhanced polyfunctional cytokine production. Moreover, fate mapping showed that a substantial fraction of the tertiary effector-memory CD8+ T cells developed from remigrated TRM cells. Thus, repeated booster vaccinations quantitatively and qualitatively improve the CD8+ T cell response leading to protection against otherwise lethal SARS-CoV-2 infection. SummaryA third dose with a single T cell epitope-vaccine promotes a strong increase in tissue-resident memory CD8+ T cells and fully protects against SARS-CoV-2 infection, while single B cell epitope-eliciting vaccines are unable to provide protection.

immunology↗

bioRxiv2021

Turnover of MCMV-expanded CD8+ T-cells is similar to that of memory phenotype T-cells and independent of the magnitude of the response

The potential of memory T-cells to provide protection against re-infection is beyond question. Yet, it remains debated whether long-term T-cell memory is due to long-lived memory cells. There is ample evidence that blood-derived memory phenotype CD8+ T-cells maintain themselves through cell division, rather than through longevity of individual cells. It has recently been proposed, however, that there may be heterogeneity in the lifespans of memory T-cells, depending on factors such as exposure to cognate antigen. Cytomegalovirus (CMV) infection induces not only conventional, contracting T-cell responses, but also inflationary CD8+ T-cell responses, which are maintained at unusually high numbers, and are even thought to continue to expand over time. It has been proposed that such inflating T-cell responses result from the accumulation of relatively long-lived CMV-specific memory CD8+ T-cells. Using in vivo deuterium labelling and mathematical modelling, we found that the average production rates and expected lifespans of mouse CMV-specific CD8+ T-cells are very similar to those of bulk memory-phenotype CD8+ T-cells. Even CMV-specific inflationary CD8+ T-cell responses that differ three-fold in size, were found to turn over at similar rates.

immunology↗

bioRxiv2021

Revealing the host antiviral protein ZAP-S as an inhibitor of SARS-CoV-2 programmed ribosomal frameshifting

Programmed ribosomal frameshifting (PRF) is a fundamental gene expression event in many viruses including SARS-CoV-2, which allows production of essential structural and replicative enzymes from an alternative reading frame. Despite the importance of PRF for the viral life cycle, it is still largely unknown how and to what extent cellular factors alter mechanical properties of frameshifting RNA molecules and thereby impact virulence. This prompted us to comprehensively dissect the interplay between the host proteome and the SARS-CoV-2 frameshift element. Here, we reveal that zinc-finger antiviral protein (ZAP-S) is a direct and specific regulator of PRF in SARS-CoV-2 infected cells. ZAP-S overexpression strongly impairs frameshifting and viral replication. Using in vitro ensemble and single-molecule techniques, we further demonstrate that ZAP-S directly interacts with the SARS-CoV-2 RNA and ribosomes and interferes with the folding of the frameshift RNA. Together these data illuminate ZAP-S as de novo host-encoded specific inhibitor of SARS-CoV-2 frameshifting and expand our understanding of RNA-based gene regulation.

molecular biology↗

bioRxiv2021

Antiviral activity of influenza A virus defective interfering particles against SARS-CoV-2 replication in vitro through stimulation of innate immunity

SARS-CoV-2 causing COVID-19 emerged in late 2019 and resulted in a devastating pandemic. Although the first approved vaccines were already administered by the end of 2020, worldwide vaccine availability is still limited. Moreover, immune escape variants of the virus are emerging against which the current vaccines may confer only limited protection. Further, existing antivirals and treatment options against COVID-19 only show limited efficacy. Influenza A virus (IAV) defective interfering particles (DIPs) were previously proposed not only for antiviral treatment of the influenza disease but also for pan-specific treatment of interferon (IFN)-sensitive respiratory virus infections. To investigate the applicability of IAV DIPs as an antiviral for the treatment of COVID-19, we conducted in vitro co-infection experiments with cell culture-derived DIPs and the IFN-sensitive SARS-CoV-2 in human lung cells. We show that treatment with IAV DIPs leads to complete abrogation of SARS-CoV-2 replication. Moreover, this inhibitory effect was dependent on janus kinase/signal transducers and activators of transcription (JAK/STAT) signaling. Further, our results suggest boosting of IFN-induced antiviral activity by IAV DIPs as a major contributor in suppressing SARS-CoV-2 replication. Thus, we propose IAV DIPs as an effective antiviral agent for treatment of COVID-19, and potentially also for suppressing the replication of new variants of SARS-CoV-2.

microbiology↗

bioRxiv2021

Differential serotonin uptake mechanisms at the human maternal-fetal interface

Mechanisms regulating serotonin (5-HT) homeostasis at the maternal-fetal interface are important for proper placental functioning and fetal (neuro)development. Here we studied 5-HT uptake mechanisms in human primary trophoblasts, feto-placental endothelial cells and cord blood platelets, all isolated directly after birth. Trophoblasts and cord blood platelets demonstrated high-affinity 5-HT uptake with similar Michaelis constant (Km) values (0.60{+/-}0.27 and 0.65{+/-}0.18 M, respectively). In contrast, feto-placental endothelial cells displayed saturation kinetics only over the low-affinity range of 5-HT concentrations (Km=782{+/-}218 M). 5-HT uptake into trophoblasts was inhibited by various psychotropic drugs targeting high-affinity serotonin transporter (SERT), and into feto-placental endothelial cells by an inhibitor of low-affinity transporters. SERT mRNAs were abundant in trophoblasts, but sparse in feto-placental endothelial cells; the opposite was found for transcripts of the low-affinity plasma membrane monoamine transporter (PMAT). These results show for the first time the presence of functional 5-HT uptake systems in feto-placental endothelial cells and fetal platelets, cells in direct contact with the fetal blood plasma. Data also emphasize sensitivity of 5-HT transport into trophoblasts, cells facing maternal blood, to various psychotropic drugs. The multiple, high- and low-affinity, systems present for cellular 5-HT uptake highlight the importance of maintaining 5-HT homeostasis at the maternal-fetal interface.

cell biology↗

bioRxiv2020

A SARS-CoV-2 neutralizing antibody selected from COVID-19 patients by phage display is binding to the ACE2-RBD interface and is tolerant to known RBD mutations

The novel betacoranavirus SARS-CoV-2 causes a form of severe pneumonia disease, termed COVID-19 (coronavirus disease 2019). Recombinant human antibodies are proven potent neutralizers of viruses and can block the interaction of viral surface proteins with their host receptors. To develop neutralizing anti-SARS-CoV-2 antibodies, antibody gene libraries from convalescent COVID-19 patients were constructed and recombinant antibody fragments (scFv) against the receptor binding domain (RBD) of the S1 subunit of the viral spike (S) protein were selected by phage display. The selected antibodies were produced in the scFv-Fc format and 30 showed more than 80% inhibition of spike (S1-S2) binding to cells expressing ACE2, assessed by flow cytometry screening assay. The majority of these inhibiting antibodies are derived from the VH3-66 V-gene. The antibody STE90-C11 showed a sub nM IC50 in a plaque-based live SARS-CoV-2 neutralization assay. The in vivo efficacy of the antibody was demonstrated in the Syrian hamster and in the hACE2 mice model using a silenced human IgG1 Fc part. The crystal structure of STE90-C11 Fab in complex with SARS-CoV-2-RBD was solved at 2.0 [A] resolution showing that the antibody binds at the same region as ACE2 to RBD. The binding and inhibtion of STE90-C11 is not blocked by many known RBD mutations including N439K, L452R, E484K or L452R+E484Q (emerging B.1.617). STE90-C11 derived human IgG1 with Fc{gamma}R silenced Fc (COR-101) is currently undergoing Phase Ib/II clinical trials for the treatment of moderate to severe COVID-19. In BriefHuman antibodies were selected from convalescent COVID-19 patients using antibody phage display. The antibody STE90-C11 is neutralizing authentic SARS-CoV-2 virus in vitro and in vivo and the crystal structure of STE90-C11 in complex with SARS-CoV-2-RBD revealed that this antibody is binding in the RBD-ACE2 interface. S1 binding of STE90-C11 and inhibition of ACE2 binding is not blocked by many known RBD mutations.

bioengineering↗

bioRxiv2020

SARS-CoV-2 Quasispecies Mediate Rapid Virus Evolution and Adaptation

Since the pandemic spread of SARS-CoV-2, the virus has exhibited remarkable genome stability, but recent emergence of novel variants show virus evolution potential. Here we show that SARS-CoV-2 rapidly adapts to Vero E6 cells that leads to loss of furin cleavage motif in spike protein. The adaptation is achieved by asymptotic expansion of minor virus subpopulations to dominant genotype, but wildtype sequence is maintained at low percentage in the virus swarm, and mediate reverse adaptation once the virus is passaged on human lung cells. The Vero E6-adapted virus show defected cell entry in human lung cells and the mutated spike variants cannot be processed by furin or TMPRSS2. However, the mutated S1/S2 site is cleaved by cathepsins with higher efficiency. Our data show that SARS-CoV-2 can rapidly adapt spike protein to available proteases and advocate for deep sequence surveillance to identify virus adaptation potential and novel variant emergence. Significance StatementRecently emerging SARS-CoV-2 variants B1.1.1.7 (UK), B.1.351 (South Africa) and B.1.1.248 (Brazil) harbor spike mutation and have been linked to increased virus pathogenesis. The emergence of these novel variants highlight coronavirus adaptation and evolution potential, despite the stable consensus genotype of clinical isolates. We show that subdominant variants maintained in the virus population enable the virus to rapidly adapt upon selection pressure. Although these adaptations lead to genotype change, the change is not absolute and genome with original genotype are maintained in virus swarm. Thus, our results imply that the relative stability of SARS-CoV-2 in numerous independent clinical isolates belies its potential for rapid adaptation to new conditions.

microbiology↗

bioRxiv2020

SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface

COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a novel betacoronavirus discovered in December 2019 and closely related to the SARS coronavirus (CoV). Both viruses use the human ACE2 receptor for cell entry, recognizing it with the Receptor Binding Domain (RBD) of the S1 subunit of the viral spike (S) protein. The S2 domain mediates viral fusion with the host cell membrane. Experience with SARS and MERS coronaviruses has shown that potent monoclonal neutralizing antibodies against the RBD can inhibit the interaction with the virus cellular receptor (ACE2 for SARS) and block the virus cell entry. Assuming that a similar strategy would be successful against SARS-CoV-2, we used phage display to select from the human naive universal antibody gene libraries HAL9/10 anti-SARS-CoV-2 spike antibodies capable of inhibiting interaction with ACE2. 309 unique fully human antibodies against S1 were identified. 17 showed more than 75% inhibition of spike binding to cells expressing ACE2 in the scFv-Fc format, assessed by flow cytometry and several antibodies showed even an 50% inhibition at a molar ratio of the antibody to spike protein or RBD of 1:1. All 17 scFv-Fc were able to bind the isolated RBD, four of them with sub-nanomolar EC50. Furthermore, these scFv-Fc neutralized active SARS-CoV-2 virus infection of VeroE6 cells. In a final step, the antibodies neutralizing best as scFv-Fc were converted into the IgG format. The antibody STE73-2E9 showed neutralization of active SARS-CoV-2 with an IC50 0.43 nM and is binding to the ACE2-RBD interface. Universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovered patients in a pandemic situation.

biochemistry↗

bioRxiv2018

Off-target effects of Cre recombinase reveal limits of adoptive T cell transfers and persistent proliferation of effector CD8 T-cells

Effector-memory T-cells (TEM) are assumed to be short-lived cells that poorly proliferate upon antigenic restimulation, thus depending on central-memory T-cells (TCM) to replenish their numbers during homeostasis, largely depending on adoptive transfer evidence. Here we analyzed T cells in their natural environment and observed robust long-term in vivo cycling within the TEM subset that was stronger than the one in the TCM subset. Murine Cytomegalovirus (MCMV) induces inflationary TEM responses that remain high during latency. We analyzed Ki67 expression during acute and latent MCMV infection and found Ki67hiBcl2lo TEM in latently infected mice, arguing for antigen-driven TEM proliferation. TEM acquired deuterium more rapidly than TCM in an in vivo labeling experiment, and were replenished more rapidly than TCM after memory depletion, suggesting that TEM cycle faster than TCM. We depleted selectively the proliferating T-cells by Cre-overinduction, which resulted in a selective loss of Ki67hiBCl2lo effector T-cells, and an increase in the death of TEM in the spleen, while it hardly affected the TCM subset, arguing for robust proliferation of TEM in the spleen. On the other hand, TEM homing to the spleen upon adoptive transfer was substantially poorer than TCM, explaining the previously reported expansions of TCM, but not TEM, upon transfer. In conclusion, our data suggest that memory inflation is maintained by proliferation of antigen-specific TEM, rather than by continued expansion and differentiation of TCM.\n\nAuthor SummaryThe naive T cell population consists of T cells that have the potential to recognize millions of different pathogens. Upon infection, naive T cells that recognize the pathogen expand, and differentiate into effector T cells that eliminate infected cells. Once the infection is contained, the T cell pool contracts and only a small population of central memory T cells remains that can expand quickly upon re-infection. Cytomegaloviruses cause persistent infections that are not cleared from the organism after the initial immune response. In infected individuals a pool of CMV-specific effector memory T cells dominates the immune system in a phenomenon called memory inflation. Previous research using the transfer of central memory or effector memory T cells from CMV-infected mice into mice with a matching infection, showed expansion of central memory T cells but not effector memory T cells. Here we show that effector memory T cells have a reduced capacity to home into lymphoid organs, where T cell activation takes place, compared to central memory T cells. Using methods that do not interfere with T cell differentiation and homing, we show that effector memory T cells are proliferating during the persistent phase of CMV infection, significantly contributing to the upkeep of the inflationary population.

immunology↗