20 research outputs found
Monitoring the T-Cell Receptor Repertoire at Single-Clone Resolution
The adaptive immune system recognizes billions of unique antigens using highly variable T-cell receptors. The Ī±Ī² T-cell receptor repertoire includes an estimated 10(6) different rearranged Ī² chains per individual. This paper describes a novel micro-array based method that monitors the Ī² chain repertoire with a resolution of a single T-cell clone. These T-arrays are quantitative and detect T-cell clones at a frequency of less than one T cell in a million, which is 2 logs more sensitive than spectratyping (immunoscope), the current standard in repertoire analysis. Using T-arrays we detected CMV-specific CD4+ and CD8+ T-cell clones that expanded early after viral antigen stimulation in vitro and in vivo. This approach will be useful in monitoring individual T-cell clones in diverse experimental settings, and in identification of T-cell clones associated with infectious disease, autoimmune disease and cancer
Transcriptional profiling of human VĪ“1 TĀ cells reveals a pathogen-driven adaptive differentiation program
Ī³Ī“ T cells are generally considered innate-like lymphocytes, however, an āadaptive-likeā Ī³Ī“ compartment has now emerged. To understand transcriptional regulation of adaptive Ī³Ī“ T cell immunobiology, we combined single-cell transcriptomics, T cell receptor (TCR)-clonotype assignment, ATAC-seq, and immunophenotyping. We show that adult VĪ“1+ T cells segregate into TCF7+LEF1+Granzyme Bneg (Tnaive) or T-bet+Eomes+BLIMP-1+Granzyme B+ (Teffector) transcriptional subtypes, with clonotypically expanded TCRs detected exclusively in Teffector cells. Transcriptional reprogramming mirrors changes within CD8+ Ī±Ī² T cells following antigen-specific maturation and involves chromatin remodeling, enhancing cytokine production and cytotoxicity. Consistent with this, in vitro TCR engagement induces comparable BLIMP-1, Eomes, and T-bet expression in naive VĪ“1+ and CD8+ T cells. Finally, both human cytomegalovirus and Plasmodium falciparum infection in vivo drive adaptive VĪ“1 T cell differentiation from Tnaive to Teffector transcriptional status, alongside clonotypic expansion. Contrastingly, semi-invariant VĪ³9+VĪ“2+ T cells exhibit a distinct āinnate-effectorā transcriptional program established by early childhood. In summary, adaptive-like Ī³Ī“ subsets undergo a pathogen-driven differentiation process analogous to conventional CD8+ T cells
Differential usage of cellular niches by cytomegalovirus versus EBV- and influenza virus-specific CD8+ T cells
Immunological memory provides long-term protection against reinfection or reactivation of pathogens. Murine memory T cell populations may be compressed following infections with new pathogens. Humans have to retain memory T cells directed against a variety of microbes for many decades. Under these circumstances, the effect of pathogens that mount robust T cell reactivity on the pre-existing memory directed against unrelated microbes is unknown. In this study, we studied peripheral blood memory CD8+ T cells directed against different viruses following primary CMV infection in renal transplant recipients. The entrance of CMV-specific CD8+ T cells expanded the Ag-primed CD8+ T cell compartment rather than competing for space with pre-existing memory T cells specific for persistent or cleared viruses. Neither numbers nor phenotype of EBV- or influenza-specific CD8+ T cells was altered by primary CMV infection. CMV-specific CD8+ T cells accumulated over time, resulting in increased total CD8+ T cell numbers. Additionally, they acquired a highly differentiated cytolytic phenotype that was clearly distinct from EBV- or influenza-reactive T cells. Thus, the human immune system appears to be flexible and is able to expand when encountering CMV. In view of the phenotypic differences between virus-specific T cells, this expansion may take place in cellular niches different from those occupied by EBV- or influenza-specific T cells, thereby preserving immunity to these pathogens
The T-array protocol.
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<b>(A)</b> During development, VDJ recombination causes enormous variability in TCRĪ² chain by randomly selecting different combinations of 23 V, 2 D, and 13 J gene segments, by nucleotide insertion (), and by nucleotide deletion from V (), D, and J () genes.</p>
<p>This results in a diversity of an estimated 10<sup>6</sup> different Ī² chains per individual.</p>
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<b>(B)</b> N-deletion causes shortening of the VĪ² and JĪ² segments.</p>
<p>The number of nucleotides deleted from VĪ² and JĪ² germline DNA is limited.</p>
<p>N-deletion of 192 published TCRĪ² mRNAs was determined.</p>
<p>The figure shows the cumulative percentage of CDR3Ī²s for the number of nucleotides deleted.</p>
<p>TCRĪ²'s with n nucleotides deleted represent approximately 10% of the repertoire if nā=ā0 to 6, and 5%, if nā=ā7 to 9.</p>
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<b>(C)</b> The T-array protocol: <b>(C1)</b> cDNA from T-cells is generated.</p>
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<b>(C2)</b> CDR3Ī² regions are PCR amplified using biotinylated VĪ²-specific () or VĪ²-generic primers (not shown here).</p>
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<b>(C3)</b> Biotinylated strands are removed after alkaline denaturation using streptavidin-coated beads.</p>
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<b>(c4)</b> Single-strands of polyclonal TCRs are aliquoted and hybridized to fluorescently labeled annealers () complementary to the NDN-adjacent end of a JĪ² gene.</p>
<p>A specific number of JĪ²-gene nucleotides (n) is deleted for each annealer, accounting for N-deletion during the VDJ recombination process.</p>
<p>Insert (C4): Each annealer will hybridize to TCRĪ² rearrangements where n nucleotides are deleted from the JĪ²-germline gene segment (<b>C4A</b>) or where less than n nucleotides are deleted (<b>C4B</b>).</p>
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<b>(C5)</b> The annealer-hybridized fractions are loaded on universal hexamer arrays for <b>(C6)</b> T-cell-clone-specific ligation and, <b>(C7)</b> subsequently washed, scanned and analyzed.</p></div
Comparison of the diversity of the human TCRb repertoire and the sensitivity of various methods for repertoire analysis.
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<b>(A)</b> The human T-cell repertoire contains an estimated 10<sup>6</sup> rearrangements per individual (<i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000055#pone.0000055-Arstila1" target="_blank">ref. 2</a></i>).</p>
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<b>(B)</b> The sensitivity of VĪ²/CĪ² immunoscope is approximately 2 in 10<sup>4</sup> (<i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000055#pone.0000055-Hohlfeld1" target="_blank">ref. 15</a> and </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000055#pone-0000055-g003" target="_blank"><i>Fig. 3A</i></a>).</p>
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<b>(C)</b> Based on the number of JĪ² primers, it is estimated that VĪ²/JĪ² immunoscope is 12-fold more sensitive than VĪ²-CĪ² immunoscope.</p>
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<b>(D,E)</b> The sensitivity by which TCR rearrangements are picked by indivual cloning, depends on the number of clones sequenced (<i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000055#pone.0000055-Betts1" target="_blank">ref. 5</a>ā<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000055#pone.0000055-Rohrlich1" target="_blank">9</a></i>).</p>
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<b>(F)</b> The sensitivity of the T-array is approximately 1 in 10<sup>6</sup> rearrangements (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000055#pone-0000055-g003" target="_blank"><i>Fig. 3C</i></a>).</p></div
Ligation experiments with Jurkat CDR3Ī² amplicons.
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<b>(A)</b> VDJ rearrangement of Jurkat TCRĪ².</p>
<p>In this CDR3 sequence, there are 3 and 0 nucleotides deleted form the germline VĪ² and JĪ², respectively.</p>
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<b>(BāE)</b> Capillary-electrophoresis chromatograms showing length and signal of fluorescent annealers ().</p>
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<b>(B)</b> A hexamer (GTTCGG) complementary to the Jurkat 3-end NDN sequence elongated (<b>*</b>) the JĪ²1-2-specific annealer (ā¦).</p>
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<b>(C)</b> The non-complementary 5ā²-end hexamer failed to cause elongation.</p>
<p>Black peaks: Cy5 signal; Red peaks: FAM signal of size standards.</p>
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<b>(D, E)</b> Similarly, a Cy5-labeled oligonucleotide complementary to the 3ā² coding end of VĪ²12, was elongated (<b>*</b>) only in the presence of the hexamer GTCGAG which is complementary to the 5ā²-end of the NDN sequence.</p>
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<b>(F)</b> Detail of universal hexamer array after ligation of a Cy5-labeled oligonucleotide complementary to the 5ā² coding end of JĪ²1-2 in the presence of the antisense strand template of Jurkat CDR3Ī².</p>
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<b>(G)</b> Signal intensities of all 4096 spots of the same array experiment.</p>
<p>The arrow in (F) and (G) indicates the microarray spot with sequence GTTCGG.</p>
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<b>(H)</b> List of 20 strongest spots with their hexamer sequence and Cy5-fluorescent signal.</p></div
Clonal expansion of T-cells from a CMV<sup>+</sup> donor after antigen specific stimulation.
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<b>(panel A)</b> The fraction of antigen specific T-cell clones determined with NLVPMVATV-loaded tetramers.</p>
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<b>(panel B)</b> Spectratyping of unsorted VĪ²13<sup>+</sup> and VĪ²13<sup>+</sup>/JĪ²1-2<sup>+</sup> fraction.</p>
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<b>(panel C)</b> CDR3 sequence of the clone identified compared to the JĪ²1-2 germline sequence.</p>
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<b>(panel D)</b> T-array of unsorted VĪ²13<sup>+</sup>/JĪ²1-2<sup>+</sup> fraction.</p>
<p>The annealer oligonucleotide used in this T-array experiment had the sequence ACTATGGCTACACCTTCGGTT, allowing detection of rearrangements with 3 or less nucleotides deleted from the JĪ²1-2 germline.</p>
<p>The arrow indicates sequence CCTTTT, the first nucleotides of the NDNĪ² region of the dominant T-cell clone that was identified in this screen.</p></div
Spectratyping and T-array for Jurkat T-cell clone mixed with peripheral blood CD4<sup>+</sup> T-cells.
<div><p>Jurkat cells were added in different dilutions to a background of peripheral blood CD4<sup>+</sup> T-cells.</p>
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<b>(panel A)</b> CDR3 spectratyping with VĪ²12-specific primer.</p>
<p>The arrow indicates a length identical to 14 amino acids, which is the length of the Jurkat CDR3Ī².</p>
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<b>(panel B)</b> A detail of the T-array scans.</p>
<p>White arrows indicate hexamer sequence GTTCGG, which is complementary to the first six nucleotides Jurkat NDNĪ² region.</p>
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<b>(panel C)</b> Signal intensities of all 4096 spots of the T-array.</p>
<p>Black arrows indicate hexamer sequence GTTCGG.</p></div