TCR diversity and the development of regulatory T cells in the human thymus

A diverse T cell receptor (TCR) repertoire is essential for the adaptive immune system. T cells have to recognise and react to a huge variety of potential pathogens and at the same time maintain tolerance to self-antigens. TCR repertoire is established in the thymus during T cell development. Previous estimates of TCR diversity have studied the mature repertoire in the peripheral blood, identifying 1-3 x 106 unique TCRβ and 0.5 x 106 TCRα sequences, but the human intrathymic diversity has not been previously measured. In this thesis I provide the first estimate of the total TCR diversity in the human thymus. We have used high-throughput TCR sequencing and detected up to 10.3 x 106 unique TCRβ sequences and 3.7 x 106 TCRα sequences, which sets a new lower limit for TCR diversity. Computational estimators preseq, DivE and Chao2 produced 40 to 70 x 106 unique TCRβ sequences and 60 to 100 x 106 TCRα sequences in the human thymus. Somatic recombination produces a diverse thymic TCR repertoire that includes also the thymocytes instructed to develop into natural regulatory T cells. They are characterised by stable expression of the forkhead/winged-helix transcription factor FOXP3 and demethylation of Treg-specific-demethylated region (TSDR). Regulatory T cells develop as a separate cell lineage in the thymus and commitment occurs already at the CD4+CD8+ double positive (DP) stage. TCR-mediated signalling instructs the selection to the regulatory lineage and cytokines promote Treg development. We have used high-throughput TCR sequencing and methylation analysis of TSDR to track the timing of Treg commitment. Comparing the common TCR sequences showed that regulatory DP CD25+ and CD4SP CD25+ populations were clearly closer to each other than any other population, indicating that they are part of the common clonal and developmental pathway. The methylation analysis of TSDR in the DP and CD4+ single positive (SP) regulatory populations showed no difference between them. Thus a stable Treg phenotype can already be detected at the double positive stage. In this thesis I have studied the role of common γ chain cytokines in regulatory T cell development in the human thymus. Since thymic commitment of human Tregs begins at the double positive stage I have studied the effects and mechanisms of interleukin-2, interleukin-7 and interleukin-15 at the DP phase. Previous studies in murine models have established a two-step model. According to these studies, TCR signalling upregulates interleukin-2 receptor (IL-2R) and increases the responsiveness of developing Tregs to common γ chain cytokines that induce the expression of FOXP3. IL-7 has been considered to have a minor role in Treg development since mature peripheral Tregs express low levels of interleukin-7 receptor (IL-7R). We show that during thymic development DP FOXP3+ thymocytes upregulate IL-7R and are responsive to IL-7. At the DP stage TCR-signalling dictates Treg maturation during positive selection and IL-15 enhances Treg survival. After positive selection IL-2 and IL-7 increase the expression of FOXP3 and anti-apoptotic protein BCL-2 (B cell lymphoma 2) and promote Treg phenotype. DP FOXP3+ cells are highly susceptible to apoptosis but all three cytokines enhance their survival. Suppressing apoptosis is a key mechanism in promoting Treg development at the DP and CD4SP stage in the human thymus.Laaja ja monimuotoinen T-solureseptorien repertuaari on välttämätön osa adaptiivista immuunipuolustusta. T-solujen täytyy tunnistaa valtava kirjo erilaisia vieraita patogeeneja, mutta samaan aikaan ylläpitää toleranssia ja jättää omat kudokset rauhaan. T-solujen repertuaari syntyy kateenkorvassa. Ihmisen kateenkorvan T-solujen repertuaarin laajuutta ei ole aiemmin tutkittu. Tässä väitöskirjatyössä on ensimmäisen kerran mitattu ja arvioitu ihmisen kateenkorvan T-solureseptorien diversiteettia. Sekvensoimme 10.3 miljoonaa uniikkia T-solureseptorin beeta-ketjua ja 3.7 miljoonaa alfa-ketjua. Nämä tulokset asettavat uuden alarajan reseptorien laajuudelle ja monimuotoisuudelle ihmisessä. Käytimme lisäksi matemaattisia estimaattoreita, jotka arvioivat koko kateenkorvan diversiteetiksi 40-70 miljoonaa uniikkia beeta-ketjua ja 60-100 miljoonaa uniikkia alfa-ketjua. Kateenkorvan somaattisen rekombinaation koneisto tuottaa laajan T-solureseptorirepertuaarin, joka sisältää myös luonnollisiksi säätelijä-T-soluiksi kehittyvät tymosyytit. Luonnolliset säätelijä-T-solut ilmentävät transkriptiotekijä FOXP3:a ja niiden geeneissä TSDR-alue on pysyvästi demetyloitunut eli siitä puuttuvat geenin hiljentävät epigeneettiset merkit. Säätelijä-T-solut kehittyvät omana solulinjanaan kateenkorvassa ja valikoituvat kehitykseen jo CD4+CD8+ tuplapositiivisessa vaiheessa. Tässä työssä olemme käyttäneet T-solureseptorien repertuaarin sekvensointia sekä TSDR-alueen metylaatioanalyysia selvittääksemme säätelijä-T-solulinjalle valikoitumisen ajoitusta. Vertailimme kateenkorvassa CD25+ populaatioiden T-solurepertuaaria CD4+CD8+ tuplapositiivisessa sekä CD4-positiivisessa kehitysvaiheessa. Yhteisiä sekvenssejä vertaamalla totesimme, että nämä molemmat CD25+ ryhmät olivat repertuaariltaan selvästi lähimpinä toisiaan. Tämä osoittaa niiden olevan osa samaa säätelijä-T-solujen kehityskaarta. Populaatioissa ei ollut myöskään mitään eroa metylaatiostatuksessa. Vakaa säätelijä-T-solun fenotyyppi voidaan havaita jo tuplapositiivisessa kehitysvaiheessa. Olen tässä väitöskirjatyössä tutkinut sytokiinien roolia säätelijä-T-solujen kehityksessä ihmisen kateenkorvassa. Nämä solut valikoituvat kehityslinjalleen jo tuplapositiivisessa vaiheessa, joten olen keskittynyt interleukiini-2:n, interleukiini-7:n ja interleukiini-15:n vaikutuksiin ja mekanismeihin tässä kehitysvaiheessa. Aiemmat tutkimukset hiirimalleissa ovat luoneet perustan kahden-askeleen-mallille: T-solureseptorin kautta välittyvä signalointi lisää CD25-molekyylin määrää solujen pinnalla ja näin herkistää niitä sytokiinien vaikutukselle. Sytokiinit käynnistävät FOXP3:n ilmentymisen. Tuplapositiivisessa kehitysvaiheessa T-solureseptorin kautta välittyvät signaalit ohjaavat säätelijä-T-solujen valikoitumista positiivisessa selektiossa, ja IL-15 edistää solujen edistää solujen selviytymistä. Positiivisen selektion jälkeen IL-2 ja IL-7 lisäävät FOXP3:n ja anti-apoptoottisen BCL-2:n ilmentymistä sekä edistävät säätelijäsolun fenotyyppiä. Tuplapositiiviset FOXP3-solut ovat hyvin alttiita solukuolemalle, mutta kaikki kolme sytokiinia edistävät niiden selviytymistä. Ohjelmoidun solukuoleman ehkäiseminen onkin tärkein mekanismi, jolla sytokiinit edistävät säätelijä-T-solujen kehitystä ihmisen kateenkorvassa

Epigenetic and transcriptional analysis supports human regulatory T cell commitment at the CD4+CD8+thymocyte stage

The natural CD25 + FOXP3 + regulatory T cell (Treg) population is generated as a distinct lineage in the thymus, but the details of Treg development in humans remain unclear, and the timing of Treg commitment is also contested. Here we have analyzed the emergence of CD25 + cells at the CD4 + CD8 + double positive (DP) stage in the human thymus. We show that these cells share T cell receptor repertoire with CD25 + CD4 single-positive thymocytes, believed to be committed Tregs. They already have a fully demethylated FOXP3 enhancer region and thus display stable expression of FOXP3 and the associated Treg phenotype. Transcriptome analysis also grouped the DP CD25 + and CD4 CD25 + thymocytes apart from the CD25 - subsets. Together with earlier studies, our data are consistent with human Treg commitment already at the DP thymocyte stage. We suggest that the most important antigens and signals necessary for human Treg differentiation may be found in the thymic cortex.Peer reviewe

Human thymic T cell repertoire is imprinted with strong convergence to shared sequences

A highly diverse repertoire of T cell antigen receptors (TCR) is created in the thymus by recombination of gene segments and the insertion or deletion of nucleotides at the junctions. Using next-generation TCR sequencing we define here the features of recombination and selection in the human TCR alpha and TCR beta locus, and show that a strikingly high proportion of the repertoire is shared by unrelated individuals. The thymic TCRa nucleotide repertoire was more diverse than TCR beta, with 4.1 x 10(6) vs. 0.81 x 10(6) unique clonotypes, and contained nonproductive clonotypes at a higher frequency (69.2% vs. 21.2%). The convergence of distinct nucleotide clonotypes to the same amino acid sequences was higher in TCRa than in TCR beta repertoire (1.45 vs. 1.06 nucleotide sequences per amino acid sequence in thymus). The gene segment usage was biased, and generally all individuals favored the same genes in both TCR alpha and TCR beta loci. Despite the high diversity, a large fraction of the repertoire was found in more than one donor. The shared fraction was bigger in TCR alpha than TCR beta repertoire, and more common in in-frame sequences than in nonproductive sequences. Thus, both biases in rearrangement and thymic selection are likely to contribute to the generation of shared repertoire in humans.Peer reviewe

Characterization of human T cell receptor repertoire data in eight thymus samples and four related blood samples

T cell receptor (TCR) is a heterodimer consisting of TCR alpha and TCR beta chains that are generated by somatic recombination of multiple gene segments. Nascent TCR repertoire undergoes thymic selections where non-functional and potentially autoreactive receptors are removed. During the last years, the development of high-throughput sequencing technology has allowed a large scale assessment of TCR repertoire and multiple analysis tools are now also available. In our recent manuscript, Human thymic T cell repertoire is imprinted with strong convergence to shared sequences [1], we show highly overlapping thymic TCR repertoires in unrelated individuals. In the current Data in Brief article, we provide a more detailed characterization of the basic features of these thymic and related peripheral blood TCR repertoires. The thymus samples were collected from eight infants undergoing corrective cardiac surgery, two of whom were monozygous twins [2]. In parallel with the surgery, a small aliquot of peripheral blood was drawn from four of the donors. Genomic DNA was extracted from mechanically released thymocytes and circulating leukocytes. The sequencing of TCR alpha and TCR beta repertoires was performed at ImmunoSEQ platform (Adaptive Biotechnologies). The obtained repertoire data were analysed applying relevant features from immunoSEQ (R) 3.0 Analyzer (Adaptive Biotechnologies) and a freely available VDJTools software package for programming language R [3]. The current data analysis displays the basic features of the sequenced repertoires including observed TCR diversity, various descriptive TCR diversity measures, and V and J gene usage. In addition, multiple methods to calculate repertoire overlap between two individuals are applied. The raw sequence data provide a large database of reference TCRs in healthy individuals at an early developmental stage. The data can be exploited to improve existing computational models on TCR repertoire behaviour as well as in the generation of new models. (C) 2021 The Authors. Published by Elsevier Inc.Peer reviewe

T cell receptor diversity in the human thymus

A diverse T cell receptor (TCR) repertoire is essential for adaptive immune responses and is generated by somatic recombination of TCR alpha and TCR beta gene segments in the thymus. Previous estimates of the total TCR diversity have studied the circulating mature repertoire, identifying 1 to 3 x 10(6) unique TCR beta and 0.5 x 10(6) TCR alpha sequences. Here we provide the first estimate of the total TCR diversity generated in the human thymus, an organ which in principle can be sampled in its entirety. High-throughput sequencing of samples from four pediatric donors detected up to 10.3 x 10(6) unique TCR beta sequences and 3.7 x 10(6) TCR alpha sequences, the highest directly observed diversity so far for either chain. To obtain an estimate of the total diversity we then used three different estimators, preseq and DivE, which measure the saturation of rarefaction curves, and Chao2, which measures the size of the overlap between samples. Our results provide an estimate of a thymic repertoire consisting of 40 to 70 x 10(6) unique TCR beta sequences and 60 to 100 x 10(6) TCR alpha sequences. The thymic repertoire is thus extremely diverse. Moreover, extrapolation of the data and comparison with earlier estimates of peripheral diversity also suggest that the thymic repertoire is transient, with different clones produced at different times. (C) 2016 Elsevier Ltd. All rights reserved.Peer reviewe

Identifying the inheritable component of human thymic T cell repertoire generation in monozygous twins

We have analyzed T cell receptor repertoires in a unique set of thymus samples from a pair of monozygotic twins. While genetics affect the V(D)J rearrangement and generation of junctional sequences, the thymic selections seem largely stochastic and import no detectable inheritable effect at clonal level.Non peer reviewe

Loss-of-function mutation in IKZF2 leads to immunodeficiency with dysregulated germinal center reactions and reduction of MAIT cells

Peer reviewe

Loss-of-function mutation in IKZF2 leads to immunodeficiency with dysregulated germinal center reactions and reduction of MAIT cells

Peer reviewe

T Cell Receptor Diversity in the Human Thymus

A diverse T cell receptor (TCR) repertoire is essential for adaptive immune responses and is generated by somatic recombination of TCRβ and TCRα gene segments in the thymus. Previous estimates of the total TCR diversity have studied the circulating mature repertoire, identifying 1-3 x 106 unique TCRβ and 0.5 x 106 TCRα sequences. Here we provide the first estimate of the total TCR diversity generated in the human thymus, an organ which in principle can be sampled in its entirety. High-throughput sequencing of samples from four pediatric donors detected up to 10.3 x 106 unique TCRβ sequences and 3.7 x 106 TCRα sequences, the highest directly observed diversity so far for either chain. To obtain an estimate of the total diversity we then used three different estimators, preseq and DivE, which measure the saturation of rarefaction curves, and Chao2, which measures the size of the overlap between samples. Our results indicate that the thymic repertoire consists of 40-70 x 106 unique TCRβ sequences and 60-100 x 106 TCRα sequences. The thymic repertoire is thus extremely diverse, but the data also suggest that it is transient, with different clones produced at different times