110 research outputs found
How αβ T cells deal with induced TCRα ablation
No full textOn deletion of the gene encoding the constant region of the T cell antigen receptor (TCR) a chain in mature T cells by induced Cremediated recombination, the cells lose most of their TCR from the cell surface within 7–10 days, but minute amounts of surfacebound TCR b chains are retained for long periods of time. In a situation in which cellular influx from the thymus is blocked, TCR-deficient naı¨ve T cells decay over time, the decay rates being faster for CD81 cells (t1/2 ' 16 days) than for CD41 cells (t1/2 ' 46 days).TCR1 naı¨vecellsareeithermaintained(CD81)ordecaymore slowly (CD41; t1/2 ' 78 days.) Numbers of TCR-deficient memory T cells decline very slowly (CD81 cells; t1/2 ' 52 days) or not at all (CD41 cells), but at the population level, these cells fail to expand as their TCR1 counterparts do. Together with earlier data on T cell maintenanceinenvironmentslackingappropriatemajorhistocompatibility complex antigens, these data argue against the possibility that spontaneous ligand-independent signaling by the ab TCR contributes significantly to T-cell homeostasis
Adoptive transfer of cytomegalovirus-specific CTL to stem cell transplant patients after selection by HLA–peptide tetramers
No full textStem cell transplantation is used widely in the management of a range of diseases of the hemopoietic system. Patients are immunosuppressed profoundly in the early posttransplant period, and reactivation of cytomegalovirus (CMV) remains a significant cause of morbidity and mortality. Adoptive transfer of donor-derived CMV-specific CD8+ T cell clones has been shown to reduce the rate of viral reactivation; however, the complexity of this approach severely limits its clinical application. We have purified CMV-specific CD8+ T cells from the blood of stem cell transplant donors using staining with HLA–peptide tetramers followed by selection with magnetic beads. CMV-specific CD8+ cells were infused directly into nine patients within 4 h of selection. Median cell dosage was 8.6 × 103/kg with a purity of 98% of all T cells. CMV-specific CD8+ T cells became detectable in all patients within 10 d of infusion, and TCR clonotype analysis showed persistence of infused cells in two patients studied. CMV viremia was reduced in every case and eight patients cleared the infection, including one patient who had a prolonged history of CMV infection that was refractory to antiviral therapy. This novel approach to adoptive transfer has considerable potential for antigen-specific T cell therapy
Visualization and phenotyping of proinflammatory antigen-specific T cells during collagen-induced arthritis in a mouse with a fixed collagen type II-specific transgenic T-cell receptor beta-chain
No full textIntroduction: The Vbeta12-transgenic mouse was previously generated to investigate the role of antigen-specific T cells in collagen-induced arthritis (CIA), an animal model for rheumatoid arthritis. This mouse expresses a transgenic collagen type II (CII)-specific T-cell receptor (TCR) beta-chain and consequently displays an increased immunity to CII and increased susceptibility to CIA. However, while the transgenic Vbeta12 chain recombines with endogenous alpha-chains, the frequency and distribution of CII-specific T cells in the Vbeta12-transgenic mouse has not been determined. The aim of the present report was to establish a system enabling identification of CII-specific T cells in the Vbeta12-transgenic mouse in order to determine to what extent the transgenic expression of the CII-specific beta-chain would skew the response towards the immunodominant galactosylated T-cell epitope and to use this system to monitor these cells throughout development of CIA. Methods: We have generated and thoroughly characterized a clonotypic antibody, which recognizes a TCR specific for the galactosylated CII(260-270) peptide in the Vbeta12-transgenic mouse. Hereby, CII-specific T cells could be quantified and followed throughout development of CIA, and their phenotype was determined by combinatorial analysis with the early activation marker CD154 (CD40L) and production of cytokines. Results: The Vbeta12-transgenic mouse expresses several related but distinct T-cell clones specific for the galactosylated CII peptide. The clonotypic antibody could specifically recognize the majority (80%) of these. Clonotypic T cells occurred at low levels in the naïve mouse, but rapidly expanded to around 4% of the CD4+ T cells, whereupon the frequency declined with developing disease. Analysis of the cytokine profile revealed an early Th1-biased response in the draining lymph nodes that would shift to also include Th17 around the onset of arthritis. Data showed that Th1 and Th17 constitute a minority among the CII-specific population, however, indicating that additional subpopulations of antigen-specific T cells regulate the development of CIA. Conclusions: The established system enables the detection and detailed phenotyping of T cells specific for the galactosylated CII peptide and constitutes a powerful tool for analysis of the importance of these cells and their effector functions throughout the different phases of arthritis
Decreased inflammatory cytokine production of antigen-specific CD4+ T cells in NMDA receptor encephalitis
No full textAnti-N-methyl-D-aspartate-receptor (NMDAR) encephalitis is the most common autoimmune encephalitis with psychosis, amnesia, seizures and dyskinesias. The disease is mediated by pathogenic autoantibodies against the NR1 subunit that disrupt NMDAR function. Antibody infusion into mouse brains can recapitulate encephalitis symptoms, while active immunization resulted also in strong T cell infiltration into the hippocampus. However, whether T cells react against NMDAR and their specific contribution to disease development are poorly understood. Here we characterized the ex vivo frequency and phenotype of circulating CD4(+) T helper (T-H) cells reactive to NR1 protein using antigen-reactive T cell enrichment (ARTE) in 24 patients with NMDAR encephalitis, 13 patients with LGI1 encephalitis and 51 matched controls. Unexpectedly, patients with NMDAR encephalitis had lower frequencies of CD154-expressing NR1-reactive T-H cells than healthy controls and produced significantly less inflammatory cytokines. No difference was seen in T cells reactive to the synaptic target LGI1 (Leucine-rich glioma-inactivated 1), ubiquitous Candida antigens or neoantigens, suggesting that the findings are disease-specific and not related to therapeutic immunosuppression. Also, patients with LGI1 encephalitis showed unaltered numbers of LGI1 antigen-reactive T cells. The data reveal disease-specific functional alterations of circulating NMDAR-reactive T-H cells in patients with NMDAR encephalitis and challenge the idea that increased pro-inflammatory NMDAR-reactive T cells contribute to disease pathogenesis
Aire-dependent production of XCL1 mediates medullary accumulation of thymic dendritic cells and contributes to regulatory T cell development
No full textDendritic cells (DCs) in the thymus (tDCs) are predominantly accumulated in the medulla and contribute to the establishment of self-tolerance. However, how the medullary accumulation of tDCs is regulated and involved in self-tolerance is unclear. We show that the chemokine receptor XCR1 is expressed by tDCs, whereas medullary thymic epithelial cells (mTECs) express the ligand XCL1. XCL1-deficient mice are defective in the medullary accumulation of tDCs and the thymic generation of naturally occurring regulatory T cells (nT reg cells). Thymocytes from XCL1-deficient mice elicit dacryoadenitis in nude mice. mTEC expression of XCL1, tDC medullary accumulation, and nT reg cell generation are diminished in Aire-deficient mice. These results indicate that the XCL1-mediated medullary accumulation of tDCs contributes to nT reg cell development and is regulated by Aire
Mathematical modelling of T cell homeostasis
No full textT cell homeostasis describes the process through which the immune system
regulates cell survival, proliferation, differentiation and death to maintain T
cell numbers and diversity in a range of different conditions. The aim of this
thesis is to better understand how this process leads to the development of the
naive CD4+ T cell compartment during childhood. Mathematical modelling is
used in combination with experimental observations to estimate naive T cell
kinetics over the lifetime of an individual. The analysis described here shows
that post-thymic proliferation contributes more than double the number of cells
entering the pool each day from the thymus. This ratio is preserved from birth
to age 20 years; as the thymus involutes, the average time between naive T-cell
divisions in the periphery lengthens with age and the naive population is maintained
by improved naive cell survival. Thymic output is quantified from birth
to age 60 years by combining models to interpret naive T cell TRECs and Ki67
expression data. Three distinct phases of thymic T cell output are identified: (i)
increasing production from birth to age 1 year; (ii) steep decline to age 8 years;
(iii) slow decline from age 8 years onwards. Finally, the role of inter-cellular
variation in T cell residency times is explored. It is able to explain the persistence
of PTK7+ naive CD4+ T cells in thymectomised individuals. Importantly,
the model predicts the accumulation of veteran PTK7+ T cells in older individuals
and suggests that the residual population in thymectomised individuals
will also consist predominantly of veteran PTK7+ T cells. The model has implications
for the use of PTK7 as a marker of recent thymic emigration and also
naturally explains improved T cell survival in older individuals
Analyses of genetic, epigenetic and transcriptomic changes in T-prolymphocytic leukemia give insights into its leukemogenesis
No full textT-prolymphocytic leukemia (T-PLL) is an aggressive T-cell malignancy with a leukemic presentation of mature T-cells. Constitutive transcriptional activation of the oncogenes TCL1A and MTCP1 due to genomic inversions/translocations is recognized as hallmark event in T-PLL pathogenesis. The exact T-cell developmental stage in which these hallmark early events occur in T-PLL had yet to be deciphered. In addition, the non-coding landscape, and the viral pathogenic involvement in T PLL still remained unexplored. Thus, this thesis aimed firstly to track the putative cell of origin in T-PLL where the hallmark aberrations take place, secondly, it aimed to identify deregulated miRNAs in T-PLL and decipher the genetic or epigenetic causes linked with miRNA deregulation. The final aim was to determine the role of viral infection in T-PLL pathogenesis. To achieve these goals, various omic technologies ranging from genetic, epigenetic to transcriptomic analysis were used. Characterization of the hallmark oncogenic events that is, inv(14)(q11q32)/t(14;14)(q11;q32) and t(X;14)(q28;q11) was performed by using whole genome sequencing (WGS) and RNA-seq data of 17 and 11 T-PLL cases, respectively. The micro RNA (miRNA) landscape of T-PLL was analyzed by using a novel approach i.e. HTG EdgeSeq miRNA assay which is a ribonuclease protection-based assay. In addition, SNP array 6.0 was used for CNA analysis for miRNA genes. Whole genome bi-sulfite sequencing (WGBS) and histone chromatin immunoprecipitation sequencing (ChIP-seq) data of T-PLL cases and non-malignant T-cell populations were extracted from publicly available datasets. Finally, the viral landscape in T PLL was analyzed using WGS and RNA-seq data and the Kranken 2.0 software. In the first part of the thesis, using WGS data of 17 T-PLL cases the breakpoints of the hallmark aberrations in T PLL were found to be located upstream of TCL1A for cases with inv(14) and downstream of TCL1A/MTCP1 in T-PLL cases with t(14;14) and t(X;14), respectively. Moreover, RNA-seq data of 11 T-PLL cases revealed overexpression of the oncogenes TCL1A and MTCP1 in the cases harboring inv(14)/t(14;14) and t(X;14) respectively. Analyzing the junctional sequences of TRA and TCL1A/MTCP1 fusions all 17 breakpoints were found to be located at the RSS suggesting RAG1/RAG2 involvement in the process of oncogenic rearrangement at the TRA/D locus. In addition to the canonical recombination signal sequences (RSS), cryptic RSS (cRSS) was detected in three cases. It was discovered that the illegitimate TRA/TRD translocations/inversions occur at first when the TRA locus becomes accessible for rearrangement. Consequently, the T-PLL (primed) cells use the most proximal TRAV/J gene segments, just before physiologic TCL1A expression is silenced and physiological rearrangements on the second allele take place with the usage of distal or central gene segments. No mutations were detected in the normal TRA sequence confirming the functionality of the TRA protein expression. Therefore, in the first part of the thesis the transitioning stage between CD4 immature single positive T-cell to early double positive stage was found to be the time point when hallmark aberration occurs in T-PLL. The cell carrying such an oncogenic event continues maturation and rearranges the second TRA allele to achieve a functional T-cell receptor. Consequently, RAG and DNTT expression is silenced which is accordance with the mature T cell phenotype at presentation of T-PLL. The second part of the thesis is published in Genes, Chromosomes and Cancer. In this part, using ribonuclease protection-based assay, 111 of 2083 miRNAs were found to be differentially expressed in T-PLL as compared to the non-malignant T-cells. 33 of these 111 miRNAs were found to be a part of miRNA clusters and 11 of the 111 miRNAs belonged to regions of genomic gains and losses in T-PLL. No evidence of single nucleotide variations (SNVs) and indels in the mRNA genes was found indicating a relation only between copy number alterations (CNA) and miRNA deregulation. The genomic loci of the four topmost deregulated miRNAs miR-200c-141 cluster, miR-324 and miR-618 contained differentially methylated regions. Hypomethylated differentially methylated regions (DMRs) were detected in T-PLL as compared to the non-malignant T-cells at the genomic loci of the miR-200c-141 cluster and miR-324 which were moreover upregulated in T-PLL. On contrary, hypermethylated DMRs were found at the miR-618 genomic locus which was downregulated in T-PLL. This indicates a link between epigenetic factors and expression of miRNAs in T PLL. A concise set of deregulated miRNAs in T-PLL was identified, several of which were reported to be oncogenic as per previous reports. The final part of thesis is published in Leukemia and Lymphoma [160]. In this final part, the WGS and RNA-seq data of T-PLL cases were analyzed for detecting sequences of viral pathogens. The highest number of genomic hits (n=570) derived from Epstein-Barr virus (EBV) were observed in 3/17 T-PLL samples and a non-tumor control sample. PCR based verification confirmed EBV in one non-tumor T-PLL sample with highest detected viral reads. Moreover, sequences from various non-oncogenic viruses, like Roseolovirus or cytomegalovirus (CMV) were found. Transcriptomic data revealed no evidence for viruses and no viral sequence integration in the genomic sequences of T-PLL patients was detected. In conclusion, though the mining of the sequencing data detected reads derived from a heterogeneous set of viruses in both tumor and normal samples from patients with T-PLL, it does not provide evidence for a particular oncogenic virus to consistently drive the pathogenesis of T-PLL. However, this study leaves the question open for a “hit-and-run hypothesis”. Overall, this study uncovers changes in the genomic, transcriptomic, and epigenetic landscapes in T-PLL and provides a comprehensive overview on the molecular events underlying T-PLL pathogenesis which could prove to be crucial for future targeted therapeutic strategies.
T-prolymphocytic leukemia (T-PLL) is an aggressive T-cell malignancy with a leukemic presentation of mature T-cells. Constitutive transcriptional activation of the oncogenes TCL1A and MTCP1 due to genomic inversions/translocations is recognized as hallmark event in T-PLL pathogenesis. The exact T-cell developmental stage in which these hallmark early events occur in T-PLL had yet to be deciphered. In addition, the non-coding landscape, and the viral pathogenic involvement in T PLL still remained unexplored. Thus, this thesis aimed firstly to track the putative cell of origin in T-PLL where the hallmark aberrations take place, secondly, it aimed to identify deregulated miRNAs in T-PLL and decipher the genetic or epigenetic causes linked with miRNA deregulation. The final aim was to determine the role of viral infection in T-PLL pathogenesis. To achieve these goals, various omic technologies ranging from genetic, epigenetic to transcriptomic analysis were used. Characterization of the hallmark oncogenic events that is, inv(14)(q11q32)/t(14;14)(q11;q32) and t(X;14)(q28;q11) was performed by using whole genome sequencing (WGS) and RNA-seq data of 17 and 11 T-PLL cases, respectively. The micro RNA (miRNA) landscape of T-PLL was analyzed by using a novel approach i.e. HTG EdgeSeq miRNA assay which is a ribonuclease protection-based assay. In addition, SNP array 6.0 was used for CNA analysis for miRNA genes. Whole genome bi-sulfite sequencing (WGBS) and histone chromatin immunoprecipitation sequencing (ChIP-seq) data of T-PLL cases and non-malignant T-cell populations were extracted from publicly available datasets. Finally, the viral landscape in T PLL was analyzed using WGS and RNA-seq data and the Kranken 2.0 software. In the first part of the thesis, using WGS data of 17 T-PLL cases the breakpoints of the hallmark aberrations in T PLL were found to be located upstream of TCL1A for cases with inv(14) and downstream of TCL1A/MTCP1 in T-PLL cases with t(14;14) and t(X;14), respectively. Moreover, RNA-seq data of 11 T-PLL cases revealed overexpression of the oncogenes TCL1A and MTCP1 in the cases harboring inv(14)/t(14;14) and t(X;14) respectively. Analyzing the junctional sequences of TRA and TCL1A/MTCP1 fusions all 17 breakpoints were found to be located at the RSS suggesting RAG1/RAG2 involvement in the process of oncogenic rearrangement at the TRA/D locus. In addition to the canonical recombination signal sequences (RSS), cryptic RSS (cRSS) was detected in three cases. It was discovered that the illegitimate TRA/TRD translocations/inversions occur at first when the TRA locus becomes accessible for rearrangement. Consequently, the T-PLL (primed) cells use the most proximal TRAV/J gene segments, just before physiologic TCL1A expression is silenced and physiological rearrangements on the second allele take place with the usage of distal or central gene segments. No mutations were detected in the normal TRA sequence confirming the functionality of the TRA protein expression. Therefore, in the first part of the thesis the transitioning stage between CD4 immature single positive T-cell to early double positive stage was found to be the time point when hallmark aberration occurs in T-PLL. The cell carrying such an oncogenic event continues maturation and rearranges the second TRA allele to achieve a functional T-cell receptor. Consequently, RAG and DNTT expression is silenced which is accordance with the mature T cell phenotype at presentation of T-PLL. The second part of the thesis is published in Genes, Chromosomes and Cancer. In this part, using ribonuclease protection-based assay, 111 of 2083 miRNAs were found to be differentially expressed in T-PLL as compared to the non-malignant T-cells. 33 of these 111 miRNAs were found to be a part of miRNA clusters and 11 of the 111 miRNAs belonged to regions of genomic gains and losses in T-PLL. No evidence of single nucleotide variations (SNVs) and indels in the mRNA genes was found indicating a relation only between copy number alterations (CNA) and miRNA deregulation. The genomic loci of the four topmost deregulated miRNAs miR-200c-141 cluster, miR-324 and miR-618 contained differentially methylated regions. Hypomethylated differentially methylated regions (DMRs) were detected in T-PLL as compared to the non-malignant T-cells at the genomic loci of the miR-200c-141 cluster and miR-324 which were moreover upregulated in T-PLL. On contrary, hypermethylated DMRs were found at the miR-618 genomic locus which was downregulated in T-PLL. This indicates a link between epigenetic factors and expression of miRNAs in T PLL. A concise set of deregulated miRNAs in T-PLL was identified, several of which were reported to be oncogenic as per previous reports. The final part of thesis is published in Leukemia and Lymphoma [160]. In this final part, the WGS and RNA-seq data of T-PLL cases were analyzed for detecting sequences of viral pathogens. The highest number of genomic hits (n=570) derived from Epstein-Barr virus (EBV) were observed in 3/17 T-PLL samples and a non-tumor control sample. PCR based verification confirmed EBV in one non-tumor T-PLL sample with highest detected viral reads. Moreover, sequences from various non-oncogenic viruses, like Roseolovirus or cytomegalovirus (CMV) were found. Transcriptomic data revealed no evidence for viruses and no viral sequence integration in the genomic sequences of T-PLL patients was detected. In conclusion, though the mining of the sequencing data detected reads derived from a heterogeneous set of viruses in both tumor and normal samples from patients with T-PLL, it does not provide evidence for a particular oncogenic virus to consistently drive the pathogenesis of T-PLL. However, this study leaves the question open for a “hit-and-run hypothesis”. Overall, this study uncovers changes in the genomic, transcriptomic, and epigenetic landscapes in T-PLL and provides a comprehensive overview on the molecular events underlying T-PLL pathogenesis which could prove to be crucial for future targeted therapeutic strategies
Rapid detection, enrichment and propagation of specific T cell subsets based on cytokine secretion
No full textSummary
T cell lines with defined cytokine profiles are an invaluable tool for assessing the control of immune responses both in vitro and in vivo. Production of such cell lines can be complex and time-consuming. Here we present a powerful technique to assay the cytokines produced by T cells activated polyclonally or with specific antigens. This paper presents a detailed methodology for the identification and isolation of cytokine-producing T cells activated with the artificial superantigen, CytoStim, or viral and fungal antigens. These cells can be analysed for different cytokines simultaneously, or cultured further to rapidly establish T cell lines making known cytokine types. We highlight the enumeration, isolation and phenotype of interleukin-17-producing T cells, and the rapid generation of virus-specific Th1 T cell lines.</jats:p
Identification and isolation of murine antigen-reactive T cells according to CD154 expression
No full textT helper (Th) cells are central regulators of adaptive immune responses. However, the detection of the small number of Th cells specific for a particular antigen or pathogen is still a major challenge. CD154 was recently introduced as a marker for antigen-specific Th cells. To date, this technology was not applicable for mice – arguably the most important immunological model system. CD154 is difficult to detect due to its rapid removal from the cell surface upon binding to CD40 during antigen-specific activation by APC. We present an efficient strategy to block the degradation of murine CD154 by combined use of antibodies against CD40 and CD154. This strategy makes CD154 easily accessible for surface staining, which allows isolation and expansion of rare antigen specific T cells. Importantly, CD154 identified all specific T cells in strongly Th1- or Th2-polarized immune responses against pathogens like Salmonella typhimurium and Heligmosomoides polygyrus, independent of their potential to produce cytokines. We demonstrate that CD154 can in fact be used as a reliable marker for antigen-specific CD4 T cells in mice, offering a unique option to analyze, isolate and rapidly expand the entire pool of Th-cells generated during a physiological T cell response in vivo
Role and specificity of regulatory T cells during retroviral infection
No full textDuring chronic viral infection of the hematopoietic system, both virus-mediated and
immune-mediated effects may cause bone marrow dysfunction leading to cytopenias. To
study the pathological consequences of the T helper response to unresolving chronic
infection, a system was developed using infection of immunodeficient mice with Friend
virus (FV). FV is a murine retroviral complex, which causes non-cytopathic persistent
infection of hematopoietic cells. The results obtained suggested that unregulated CD4+ T
cell response to FV causes bone marrow pathology and anaemia.
Regulatory T (Treg) cells are a subset of CD4+ T cells which have been shown to
suppress immune responses and to have protective roles in other models of bone marrow
pathology. Therefore, the role of Treg cells in the model of FV-induced immune
pathology was addressed. Bone marrow pathology was triggered by local gamma
interferon (IFN-γ) production by FV-specific CD4+ T cells and was associated with
relative low numbers of Treg cells, while enrichment of the Treg cell population
protected against development of the immune pathology, by suppressing IFN-γ
production by pathogenic CD4+ T cells.
The specificity of Treg cells is still a matter of controversy, with studies suggesting they
are mainly self-reactive while other studies indicate they can be reactive to foreign
antigens. The issue of Treg cell specificity was addressed in the FV-induced immune pathology model. Analysis of mice transgenically expressing the TCRβ chain of a FVspecific
CD4+ T cell clone, which harbor a polyclonal TCR repertoire with increased
frequency of FV-specific CD4+ T cells, indicated that the TCR repertoire of Treg cells in
virus-naïve mice was virtually devoid of FV-specific clones. Moreover, FV infection did
not cause expansion of a small number of virus-specific Treg cells or conversion of virusspecific
effector T cells into Forkhead box P3 (FoxP3)-expressing Treg cells.
Importantly, pathogenic CD4+ T cells and Treg cells differed dramatically in their
requirements for direct recognition of viral antigens, since bone marrow pathology driven
by FV-specific TCRβ-transgenic CD4+ T cells was efficiently suppressed by virusnonspecific
Treg cells. Therefore, protection from bone marrow pathology in chronic
viral infections may be provided by sufficient numbers of polyclonal Treg cells
- …
