Repetitive pathogen exposure leads to the dominant outgrowth of T cell clones with high T cell receptor (TCR) affinity to the relevant pathogen-associated antigens. However, low-affinity clones are ...also known to expand and form immunological memory. While these low-affinity clones contribute less immunity to the original pathogen, their role in protection against pathogens harboring immune escape mutations remains unclear. Based on identification of the TCR repertoire and functionality landscape of naive epitope-specific CD8+ T cells, we reconstructed defined repertoires that could be followed as polyclonal populations during immune responses in vivo. We found that selective clonal expansion is governed by clear TCR avidity thresholds. Simultaneously, initial recruitment of broad TCR repertoires provided a polyclonal niche from which flexible secondary responses to mutant epitopes could be recalled. Elucidating how T cell responses develop “from scratch” is informative for the development of enhanced immunotherapies and vaccines.
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•TCR avidity distribution in naive donors is highly skewed•Clonal T cell expansion upon infection is governed by clear TCR avidity thresholds•The cross-reactivity landscape of single TCRs correlates with functional avidity•Recruitment of broad TCR repertoires enables flexible immune responses
High-avidity CD8+ T cells provide protective immunity. However, their recruitment into immune responses alongside lower avidity T cells is not well understood. Straub et al. here show that rare high-avidity clones are selectively expanded from naive precursor repertoires with heterogeneous functionalities. Such polyclonal recruitment enables flexible immune responses against heterologous re-infection.
The in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells ...require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for 'reverse phenotyping'. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.
Summary Dendritic cells phagocytose pathogens leading to maturation and cross-presentation on MHC class I. We found that the efficiency of cross-priming in mice after vaccination with biodegradable ...poly( d , l -lactide- co -glycolide) microspheres (MSs) was enhanced when ovalbumin was coencapsulated together with either a CpG oligonucleotide or polyI:C as compared to co-inoculation of ovalbumin-bearing MS with soluble or separately encapsulated adjuvants. A single immunization with MS containing coencaspsulated CpG and ovalbumin yielded 9% SIINFEKL/H-2Kb tetramer positive CTLs, production of IFN-γ, efficient cytolysis, and protection from vaccinia virus infection. Taken together, coencapsulation of adjuvant and antigen is an important paradigm for the generation of potent CTL responses.
Development of CD8+ T cell (CTL) immunity or tolerance is linked to the conditions during T cell priming. Dendritic cells (DCs) matured during inflammation generate effector/memory T cells, whereas ...immature DCs cause T cell deletion/anergy. We identify a third outcome of T cell priming in absence of inflammation enabled by cross-presenting liver sinusoidal endothelial cells. Such priming generated memory T cells that were spared from deletion by immature DCs. Similar to central memory T cells, liver-primed T cells differentiated into effector CTLs upon antigen re-encounter on matured DCs even after prolonged absence of antigen. Their reactivation required combinatorial signaling through the TCR, CD28, and IL-12R and controlled bacterial and viral infections. Gene expression profiling identified liver-primed T cells as a distinct Neuropilin-1+ memory population. Generation of liver-primed memory T cells may prevent pathogens that avoid DC maturation by innate immune escape from also escaping adaptive immunity through attrition of the T cell repertoire.
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► Memory-like CD8+ T cells are generated in the liver in the absence of inflammation ► An alternative pathway of T cell priming is facilitated by nonimmune cells ► Liver-primed T cells are rescued from deletion for anti-infectious immunity ► T cell priming in the liver complements conventional memory T cell generation
Development of T cell immunity and memory is believed to occur exclusively in the context of inflammation. Knolle and colleagues demonstrate the existence of an alternative pathway of memory T cell formation in the absence of inflammation. Such T cell memory was generated not by classical antigen-presenting cells, such as dendritic cells, but by organ-resident endothelial cells in the liver and contributes to infection control. Generation of liver-primed memory T cells counteracts immune escape of pathogens that circumvent induction of inflammation.
T-cell therapy of chronic hepatitis B is a novel approach to restore antiviral T-cell immunity and cure the infection. We aimed at identifying T-cell receptors (TCR) with high functional avidity that ...have the potential to be used for adoptive T-cell therapy. To this end, we cloned HLA-A*02-restricted, hepatitis B virus (HBV)-specific T cells from patients with acute or resolved HBV infection. We isolated 11 envelope- or core-specific TCRs and evaluated them in comprehensive functional analyses. T cells were genetically modified by retroviral transduction to express HBV-specific TCRs. CD8+ as well as CD4+ T cells became effector T cells recognizing even picomolar concentrations of cognate peptide. TCR-transduced T cells were polyfunctional, secreting the cytokines interferon gamma, tumor necrosis factor alpha and interleukin-2, and effectively killed hepatoma cells replicating HBV. Notably, our collection of HBV-specific TCRs recognized peptides derived from HBV genotypes A, B, C and D presented on different HLA-A*02 subtypes common in areas with high HBV prevalence. When co-cultured with HBV-infected cells, TCR-transduced T cells rapidly reduced viral markers within two days. Our unique set of HBV-specific TCRs with different affinities represents an interesting tool for elucidating mechanisms of TCR-MHC interaction and dissecting specific anti-HBV mechanisms exerted by T cells. TCRs with high functional avidity might be suited to redirect T cells for adoptive T-cell therapy of chronic hepatitis B and HBV-induced hepatocellular carcinoma.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Compared to "live" vaccines, the immunogenicity of "subunit" vaccines based on recombinant antigen (Ag) is poor, presumably because exogenous Ag fails to effectively access the endosomal ...Ag‐processing pathways of Ag‐presenting cells (APC). To overcome this limitation, we exploited biodegradable poly(lactic‐co‐glycolic) microspheres (MP) co‐entrapping Ag and Toll‐like receptor (TLR) 9 or 7 ligands as an endosomal delivery device. In vitro, microspheres were rapidly phagocytosed by APC and translocated into phago‐endosomal compartments, followed by degradation of the Ag and concurrent activation of endosomal TLR. As a consequence, full maturation of and cytokine secretion by APC as well as Ag‐cross‐presentation ensued. In vivo, "loaded" microspheres triggered clonal expansion of primary and secondary Ag‐specific CD4 and CD8 T cells. The efficacy of CD8 T cell cross‐priming was comparable to that of live vectors. The potency of T cell vaccination was demonstrated by protective and therapeutic interventions using infection‐ and tumor‐model systems. These preclinical "subunit" vaccination data thus recommend MP as a generally applicable and powerful endosomal delivery device of exogenous Ag plus TLR‐based adjuvants to vaccinate for protective and therapeutic CD4 and CD8 T cell immunity.
Deletion or Treg cell differentiation are alternative fates of autoreactive MHCII-restricted thymocytes. How these different modes of tolerance determine the size and composition of polyclonal ...cohorts of autoreactive T cells with shared specificity is poorly understood. We addressed how tolerance to a naturally expressed autoantigen of the central nervous system shapes the CD4 T cell repertoire. Specific cells in the tolerant peripheral repertoire either were Foxp3⁺ or displayed anergy hallmarks and, surprisingly, were at least as frequent as in the nontolerant repertoire. Despite this apparent lack of deletional tolerance, repertoire inventories uncovered that some T cell receptors (TCRs) were lost from the CD4 T cell pool, whereas others mediated Treg cell differentiation. The antigen responsiveness of these TCRs supported an affinity model of central tolerance. Importantly, the contribution of different diverter TCRs to the nascent thymic Treg cell population reflected their antigen reactivity rather than their frequency among precursors. This reveals a multilayered TCR hierarchy in CD4 T cell tolerance that separates deleted and diverted TCRs and assures that the Treg cell compartment is filled with cells of maximal permissive antigen reactivity.
The SARS-CoV-2 pandemic has highlighted the need to better define in-hospital transmissions, a need that extends to all other common infectious diseases encountered in clinical settings. To evaluate ...how whole viral genome sequencing can contribute to deciphering nosocomial SARS-CoV-2 transmission 926 SARS-CoV-2 viral genomes from 622 staff members and patients were collected between February 2020 and January 2021 at a university hospital in Munich, Germany, and analysed along with the place of work, duration of hospital stay, and ward transfers. Bioinformatically defined transmission clusters inferred from viral genome sequencing were compared to those inferred from interview-based contact tracing. An additional dataset collected at the same time at another university hospital in the same city was used to account for multiple independent introductions. Clustering analysis of 619 viral genomes generated 19 clusters ranging from 3 to 31 individuals. Sequencing-based transmission clusters showed little overlap with those based on contact tracing data. The viral genomes were significantly more closely related to each other than comparable genomes collected simultaneously at other hospitals in the same city (n = 829), suggesting nosocomial transmission. Longitudinal sampling from individual patients suggested possible cross-infection events during the hospital stay in 19.2% of individuals (14 of 73 individuals). Clustering analysis of SARS-CoV-2 whole genome sequences can reveal cryptic transmission events missed by classical, interview-based contact tracing, helping to decipher in-hospital transmissions. These results, in line with other studies, advocate for viral genome sequencing as a pathogen transmission surveillance tool in hospitals.
T cell ignorance is a specific form of immunological tolerance. It describes the maintenance of naivety in antigen‐specific T cells in vivo despite the presence of their target antigen. It is thought ...to mainly play a role during the steady state, when self‐antigens are presented in absence of costimulatory signals and at low density or to T cells of low affinity. In how far antigen‐specific T cells can also remain clonally ignorant to foreign antigens, presented in the inflammatory context of systemic infection, remains unclear. Using single‐cell in vivo fate mapping and high throughput flow cytometric enrichment, we find that high‐affinity antigen‐specific CD8+ T cells are efficiently recruited upon systemic infection. In contrast, most low‐affinity antigen‐specific T cells ignore the priming antigen and persist in the naïve state while remaining fully responsive to subsequent immunization with a high‐affinity ligand. These data establish the widespread clonal ignorance of low‐affinity T cells as a major factor shaping the composition of antigen‐specific CD8+ T cell responses to systemic infection.
Clonal ignorance describes the maintenance of T cell naivety despite the presence of cognate antigen in vivo. Generally this tolerance mechanism is thought to play a role only during the steady state. Instead, single‐cell fate mapping reveals that upon low‐affinity T cell receptor stimulation, the bulk of antigen‐specific CD8+ T cells remain ignorant, even in the context of systemic infection.
A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to ...unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve--especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4(high)/CD25(high)/CD45RA(high) 'regulatory T cells' and CD8(high)/CD62L(high)/CD45RA(neg) 'central memory T cells', have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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